Tri-, tetra-substituted-3-aminopyrrolidine derivative

ABSTRACT

A quinolone synthetic antibacterial agent and a therapeutic agent for an infection which exhibit broad spectrum and strong antibacterial activity for both Gram positive and Gram negative bacteria, and which are also highly safe are provided. The compound provided is represented by formula (I):

BACKGROUND OF THE INVENTION

This invention relates to a quinolone synthetic antibacterial drug whichis useful as a drug for human, animals, or fish, or as a antibacterialpreservatives.

Since discovery of norfloxacin, antibacterial activity andpharmacokinetics of quinolone synthetic antibacterial drugs (includingthose containing pyridobenzoxazine skeleton) have been greatly improved,and, today, they are used in chemotherapy for infections includingalmost all systemic infections, and a large number of drugs are inclinical use.

However, a of bacteria exhibiting low sensitivity for quinolonesynthetic antibacterial drugs have been recently increasing in itsnumber in clinical field. For example, bacteria which are resistant todrugs other than quinolone synthetic antibacterial drugs, and which alsoexhibit low sensitivity to quinolone synthetic antibacterial drugs areincreasing such as Gram positive coccus like Staphylococcus aureus(MRSA) and pneumococcus (PRSP) insensitive to β-Lactam antibiotics andenterococcus (VRE) insensitive to aminoglycoside antibacterial drugs.Accordingly, there is a strong clinical demand for a drug exhibiting animproved effectiveness to Gram positive coccus.

In the meanwhile, antibacterial activity of recently developed quinolonesynthetic antibacterial compounds are by far stronger than formerquinolone synthetic antibacterial compounds. However, many suchquinolone compound having high antibacterial activity have been reportedto produce side effects based on physiological or pharmacological actionnot observed in the former quinolone synthetic antibacterial compounds.For example, restrictions are imposed on the administration of somecompounds due to side effects such as development of abnormal bloodglucose level, cardiotoxicity, or delayed allergy, or development ofconvulsion, and development and use as a drug have been abandoned insome compounds. In other words, many compounds have been found to beinsufficient in their suitability as a drug due to the strong sideeffects despite their high antibacterial activity. Accordingly, a drugdesign methodology which is different from former compounds is requiredto thereby prevent the situation that a highly antibacterial compoundcan not be developed as a drug due to production of side effects. Inother words, a design methodology is required that is capable ofproducing a compound which has a considerably high antibacterialactivity comparable or similar to those of the conventional compounds,and at the same time, which is provided with suitability for a drug thatallows use of the compound as a drug, for example, high safety withoutside effects.

Exemplary side effects which have been reported for the quinolonesynthetic antibacterial agents include induction of convulsionassociated with concomitant, use of a nonsteroidal anti-inflammatoryagent, central action (relatively light central nerve disorders such asreeling, headache, and insomnia as well as serious side effects such asdevelopment of lethal convulsion), phototoxicity (photosensitivity),hepatotoxicity, and cardiotoxicity (an abnormality observed as anabnormality of electrocardiogram which induces lethal arrhythmia),delayed allergy, and abnormal glucose blood level (see Non-patentdocuments 1 to 3).

Of the side effects as mentioned above, significant recently reportedclinical cases involve cardiotoxicity (a heart abnormality inducinglethal arrhythmia which is observed as an abnormality ofelectrocardiogram with prolonged QT or QTc interval). Some commerciallyavailable quinolone antibacterial agents have been reported to produceclearly prolonged QT or QTc interval including some serious cases(abnormality of electrocardiogram inducing lethal arrhythmia)(Non-patent documents 1 to 3). Also reported are side effects such asinduction of rash, which is a result of delayed allergy, and abnormalblood glucose level.

Accordingly, in order to enable use the quinolone antibacterial agent asa human or animal drug, there is a demand for a quinolone syntheticantibacterial agent which is provided with an improved safety withweaker side effects such as induction of convulsion associated withconcomitant use of a nonsteroidal anti-inflammatory agent, centralaction, phototoxicity (photosensitivity), and hepatotoxicity, as well asside effects such as cardiotoxicity, delayed allergy, and abnormalglucose blood level. In other words, there is a strong demand for aquinolone compound simultaneously provided with a strong antibacterialactivity and selective toxicity.

[Patent Document 1] Japanese Patent Application Laid-Open No. (JP-A)61-282382

[Patent Document 2] JP-A 63-4526-1

[Patent Document 3] JP-A 2-231475

[Patent Document 4] JP-A 3-95176

[Non-patent Document 1] Hiroyuka Kobayashi Ed., “Clinical Applicationsof New-quinolone Agents”, Iyaku-Journal-Sha (2001)

[Non-patent Document 2] Drugs, Vol. 62, No. 1, page 13 (2002)

[Non-patent Document 3] Toxicology Letters, Vol. 127, page 269 (2002)

SUMMARY OF THE INVENTION

In view of the situation as described above, an object of the presentinvention is to provide a quinolone synthetic antibacterial agent and atherapeutic agent for an infection which exhibit broad spectrum andstrong antibacterial activity for both Gram positive and Gram negativebacteria, and which are also highly safe.

The inventors of the present invention have conducted investigation byfocusing on a compound which has 3-aminopyrrolidinyl group at position 7or equivalent position of a quinolone compound. In the course of theinvestigation, the inventors found that a quinolone compound having an3-aminopyrrolidinyl group which is tri- or tetra-substituted atpositions 3 and 4 represented by the following formula:

which has substituent which is typically an aliphatic substituent on thecarbon atom at position 3, and one or two substituents which are alsotypically an aliphatic substituent on the carbon atom at position 4 hasa broad spectrum of strong antibacterial activity for Gram-positive andGram-negative bacteria including drug resistant Gram positive cocci suchas multiple drug resistant pneumococcus having the resistance forquinolone. The inventors also found that such quinolone compound has notonly such high antibacterial activity but also lower cardiotoxicitycompared to the quinolone antibacterial drugs whose cardiotoxicity hasrecently been reported in clinical practice as a side effect of thequinolone antibacterial drugs. It was also found that this compound hasreduced risk of producing side effects such as delayed allergy andabnormal blood glucose level. It was also found that this compound hasexcellent oral absorptivity, organ permeability, and excretion rate inurine. Accordingly, the inventors of the present invention have foundthat the quinolone compound represented by formula (I) is a quinolonesynthetic antibacterial drug which has excellent drug propertiesincluding excellent antibacterial activity and high safety, and alsoexcellent pharmacokinetics. The present invention has been completed onthe bases of such findings.

Accordingly, the present invention provides a compound represented byfollowing formula (I):

or a salt or a hydrate thereof, wherein

R¹ represents hydrogen atom, an alkyl group containing 1 to 6 carbonatoms, a cycloalkyl group (cyclic alkyl group) containing 3 to 6 carbonatoms, or a substituted carbonyl group derived from an amino acid, adipeptide, or a tripeptide; the alkyl group being optionally substitutedwith a substituent selected from the group consisting of hydroxy group,amino group, halogen atom, an alkylthio group containing 1 to 6 carbonatoms, and an alkoxy group containing 1 to 6 carbon atoms;

R² represents hydrogen atom, an alkyl group containing 1 to 6 carbonatoms, or a cycloalkyl group containing 3 to 6 carbon atoms; the alkylgroup being optionally substituted with a substituent selected from thegroup consisting of hydroxy group, amino group, halogen atom, analkylthio group containing 1 to 6 carbon atoms, and an alkoxy groupcontaining 1 to 6 carbon atoms;

R³ represents an alkyl group containing 1 to 6 carbon atoms, acycloalkyl group containing 3 to 6 carbon atoms, an alkenyl groupcontaining 2 to 6 carbon atoms, or an alkynyl group containing 2 to 6carbon atoms; the alkyl group being optionally substituted with asubstituent selected from the group consisting of hydroxy group, aminogroup, halogen atom, an alkylthio group containing 1 to 6 carbon atoms,and an alkoxy group containing 1 to 6 carbon atoms;

R⁴ and R⁵ independently represent hydrogen atom, halogen atom, an alkylgroup containing 1 to 6 carbon atoms, an alkoxy group containing 1 to 6carbon atoms, an alkenyl group containing 2 to 6 carbon atoms, analkynyl group containing 2 to 6 carbon atoms, or an optionallysubstituted cycloalkyl group containing 3 to 6 carbon atoms; the alkylgroup, the alkoxy group, the alkenyl group, and the alkynyl group beingeither a straight chain or branched group; the alkyl group beingoptionally substituted with a substituent selected from the groupconsisting of hydroxy group, amino group, halogen atom, an alkylthiogroup containing 1 to 6 carbon atoms, and an alkoxy group containing 1to 6 carbon atoms; and with the proviso that R⁴ and R⁵ are notsimultaneously hydrogen atom; or

the substituents R⁴ and R⁵ combine together to form (a) a 3- to6-membered cyclic structure including the carbon atom shared by R⁴ andR⁵ to form a spirocyclic structure with the pyrrolidine ring, the thusformed spiro ring optionally containing oxygen atom or sulfur atom as aring member atom, and optionally being substituted with a halogen atomor an alkyl group containing 1 to 6 carbon atoms optionally having asubstituent; or (b) exomethylene group bonding to the pyrrolidine ringby double bond, the exomethylene group optionally having 1 or 2substituents selected from hydroxy group, amino group, halogen atom, analkylthio group containing 1 to 6 carbon atoms, and an alkoxy groupcontaining 1 to 6 carbon atoms;

R⁶ and R⁷ independently represent hydrogen atom or an alkyl groupcontaining 1 to 6 carbon atoms;

R⁸ represents a halogen-substituted alkyl group containing 1 to 6 carbonatoms, a halogen-substituted cycloalkyl group containing 3 to 6 carbonatoms, a halogen-substituted phenyl group, or a halogen-substitutedheteroaryl group;

R⁹ represents hydrogen atom, phenyl group, acetoxymethyl group, pivaloyloxymethyl group, ethoxycarbonyl group, choline group, dimethylaminoethyl group, 5-indanyl group, phthalidinyl group,5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl group, 3-acetoxy-2-oxobutyl group,an alkyl group containing 1 to 6 carbon atoms, an alkoxymethyl groupcontaining 2 to 7 carbon atoms, or a phenylalkyl group comprising analkylene group containing 1 to 6 carbon atoms and phenyl group;

X¹ represents hydrogen atom or a halogen atom; and

A represents nitrogen atom or a moiety represented by formula (II):

wherein X² represents hydrogen atom, an alkyl group containing 1 to 6carbon atoms, an alkoxy group containing 1 to 6 carbon atoms, cyanogroup, halogen atom, a halogen-substituted methyl group, or ahalogenomethoxy group; or X² and R⁸ may combine together to form acyclic structure including a part of the mother nucleus, the thus formedring optionally containing oxygen atom, nitrogen atom, or sulfur atom asa ring constituting atom, and optionally being substituted with an alkylgroup containing 1 to 6 carbon atoms optionally having a substituent.

The present invention also provides a drug containing the compoundrepresented by the formula (E) or a salt or a hydrate thereofrepresented by the formula (I) as its effective ingredient.

The present invention also provides a method for treating a disease byadministering the compound represented by the formula (I) or a salt or ahydrate thereof. The present invention also provides use of the compoundrepresented by the formula (I) or a salt or a hydrate thereof forproducing a drug.

The present invention provides a quinolone synthetic drug which hasexcellent drug properties such as strong antibacterial activity not onlyfor Gram negative bacteria but also for Gram-positive cocci which havebecome less sensitive to quinolone antibacterials, high safety, andfavorable pharmacokinetics.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing MBI action of Comparative compound 1 and thecompound of Example 9 against CYP3A4.

FIG. 2 is a graph showing therapeutic effects of Comparative compound 1and the compound of Example 9 in mouse local lung infection model byPRSP.

FIG. 3 is a view showing the results of X ray structural analysis forthe compound produced in Reference Example 107.

FIG. 4 is a graph showing therapeutic effects of Comparative compound 1and the compound of Example 9 in rat simple cystitis model by E. coli.

DISCLOSURE OF THE INVENTION

First, the substituents of the formula (I) are described.

R¹ represents hydrogen atom, an alkyl group containing 1 to 6 carbonatoms, a cycloalkyl group containing 3 to 6 carbon atoms, or asubstituted carbonyl group derived from an amino acid, a dipeptide, or atripeptide. When R¹ is an alkyl group, it may be substituted with asubstituent selected from the group consisting of hydroxy group, aminogroup, halogen atom, an alkylthio group containing 1 to 6 carbon atoms,and an alkoxy group containing 1 to 6 carbon atoms.

R² represents hydrogen atom, an alkyl group containing 1 to 6 carbonatoms, or a cycloalkyl group containing 3 to 6 carbon atoms. When R² isan alkyl group, it may be substituted with a substituent selected fromthe group consisting of hydroxy group, amino group, halogen atom, analkylthio group containing 1 to 6 carbon atoms, and an alkoxy groupcontaining 1 to 6 carbon atoms.

When R¹ or R² is an alkyl group, the alkyl group may be either astraight chain or a branched alkyl group. The alkyl group is preferablymethyl group, ethyl group, propyl group, or isopropyl group, and morepreferably, methyl group or ethyl group, and most preferably methylgroup.

When R¹ or R² is an alkyl group having hydroxy group or amino group asits substituent, the alkyl group may be any straight chain or branchedalkyl containing 1 to 6 carbon atoms, and the alkyl group is preferablysubstituted with the substituent at its terminal carbon atom. The alkylgroup having hydroxy group is preferably an alkyl group containing up to3 carbon atoms, and preferable examples include hydroxymethyl group,2-hydroxyethyl group, 2-hydroxypropyl group, and 3-hydroxypropyl group.The alkyl group having amino group is preferably an alkyl groupcontaining up to 3 carbon atoms, and preferable examples includeaminomethyl group, 2-aminoethyl group, 2-aminopropyl group, and3-aminopropyl group.

When R¹ or R² is an alkyl group having a halogen atom as itssubstituent, the alkyl group may be any of the straight chain orbranched alkyl groups containing 1 to 6 carbon atoms, and the halogenatom is preferably fluorine atom. The number of the fluorinesubstitution is not limited and the substitution may be mono- toperfluoro substitution. Exemplarily preferable substituents ashalogenated alkyl group are halogen-substituted alkyl groups includemonofluoromethyl group, difluoromethyl group, trifluoromethyl group, and2,2,2-trifluoroethyl group.

When R¹ or R² is an alkyl group having an alkylthio group or an alkoxygroup as its substituent, the alkyl group may be either a straight chainor a branched alkyl group, and the alkyl moiety of the alkylthio groupand the alkoxy group may also be either a straight chain or a branchedalkyl moiety. Exemplary alkyl groups having an alkylthio group includean alkylthiomethyl group, an alkylthioethyl group, and analkylthiopropyl group, and the alkylthio group in such groups ispreferably the one containing 1 to 3 carbon atoms. More preferable aremethylthiomethyl group, ethylthiomethyl group, and methylthioethylgroup. Exemplary alkyl groups having an alkoxy group include analkoxymethyl group, an alkoxyethyl group, and an alkoxypropyl group, andthe alkoxy group in such groups is preferably the one containing 1 to 3carbon atoms. More preferable are methoxymethyl group, ethoxymethylgroup, and methoxyethyl group.

When R¹ or R² is a cycloalkyl group, it is preferably cyclopropyl groupor cyclobutyl group, and more preferably, cyclopropyl group. Substituentof the cycloalkyl group may be one or more group selected from an alkylgroup containing 1 to 6 carbon atoms, halogen atom, amino group, andhydroxy group, and examples of the preferable substituent include methylgroup, ethyl group, fluorine atom, chlorine atom, amino group, andhydroxy group.

Preferable combination of R¹ and R² include the combination whereinR^(P) is hydrogen atom, an alkyl group, a cycloalkyl group, or asubstituted carbonyl group derived from an amino acid, a dipeptide, or atripeptide and R² is hydrogen atom. Among these, the preferablecombination is the one wherein R¹ is hydrogen atom, an alkyl group, or acycloalkyl group, and R² is hydrogen atom. The alkyl group in such caseis preferably methyl group or ethyl group, and more preferably, methylgroup. The cycloalkyl group is preferably cyclopropyl group orcyclobutyl group, and more preferably cyclopropyl group. The combinationof R¹ and R² is more preferably the combination wherein both R¹ and R²are hydrogen atom, or the combination wherein one of R¹ and R² ishydrogen atom, and the other is methyl group, ethyl group, fluoroethylgroup, or cyclopropyl group.

A quinolone derivative wherein R¹ is a substituted carbonyl groupderived from an amino acid, a dipeptide, or a tripeptide, and R² ishydrogen atom is useful as a prodrug. The amino acid, dipeptide, ortripeptide used in producing such a prodrug is the one which is capableof producing a free amine compound by cleavage in the living body of thepeptide bond between the carboxyl group and the amino group having R¹and R² bonded thereto. Examples of the substituted carbonyl group usedin producing such a prodrug include substituted carbonyl substituentsderived from an amino acid such as glycine, alanine, or aspartic acid; adipeptide constituted from glycine, alanine, or asparagine such asglycine-glycine, glycine-alanine, or alanine-alanine; and a tripeptideconstituted from glycine, alanine, or asparagine such asglycine-glycine-alanine, or glycine-alanine-alanine.

R³ represents an alkyl group containing 1 to 6 carbon atoms, acycloalkyl group containing 3 to 6 carbon atoms, an alkenyl groupcontaining 2 to 6 carbon atoms, or an alkynyl group containing 2 to 6carbon atoms. When R³ is an alkyl group, it may be optionallysubstituted with a substituent selected from the group consisting ofhydroxy group, amino group, halogen atom, an alkylthio group containing1 to 6 carbon atoms, and an alkoxy group containing 1 to 6 carbon atoms.

When R³ is an alkyl group, the alkyl group may be either a straightchain or a branched alkyl group. The alkyl group is preferably methylgroup, ethyl group, propyl group, or isopropyl group. Among these, thepreferred is methyl group or ethyl group, and the more preferred ismethyl group.

The cycloalkyl group containing 3 to 6 carbon atoms is preferablycyclopropyl group or cyclobutyl group, and more preferably cyclopropylgroup.

The alkenyl group containing 2 to 6 carbon atoms is preferably the onehaving one double bond, which is not particularly limited for itslocation. The preferred are vinyl group, propenyl group, and butenylgroup. The alkynyl group containing 2 to 6 carbon atoms is alsopreferably the one containing one triple bond, which is not particularlylimited for its location. The preferred are ethynyl group, propynyl, andbutynyl. Among those mentioned above, the preferred are vinyl group andethynyl group.

When R³ is an alkyl group, it may be optionally substituted with asubstituent selected from the group consisting of hydroxy group, aminogroup, halogen atom, an alkylthio group containing 1 to 6 carbon atoms,and an alkoxy group containing 1 to 6 carbon atoms.

When the substituent of the alkyl group is hydroxy group or amino group,the alkyl group is preferably substituted with such substituent at theterminal carbon atom. Preferable examples of the alkyl group havinghydroxy group are hydroxymethyl group, 2-hydroxyethyl group,2-hydroxypropyl group, and 3-hydroxypropyl group, and preferableexamples of the alkyl group having amino group are aminomethyl group,2-aminoethyl group, 2-aminopropyl group, and 3-aminopropyl group. Thealkyl group having the hydroxy group or the amino group is preferablymethyl group or ethyl group having such group, and more preferablymethyl group having such group, for example, hydroxymethyl group oraminomethyl group.

When the alkyl group has a halogen atom as its substituent, the alkylgroup may be any of the straight chain or branched alkyl groupscontaining 1 to 6 carbon atoms. The preferred is methyl group or ethylgroup having a halogen atom, and the more preferred is methyl grouphaving a halogen atom. Preferable halogen atom is fluorine atom. Thenumber of the fluorine substitution is not limited and the substitutionmay be mono- to perfluoro substitution. Exemplary halogen-substitutedalkyl groups include monofluoromethyl group, difluoromethyl group,trifluoromethyl group, and 2,2,2-trifluoroethyl group, and the preferredis monofluoromethyl group, difluoromethyl group, and trifluoromethylgroup.

When the substituent of the alkyl group is an alkylthio group or analkoxy group, the alkyl group may be either a straight chain or abranched alkyl group, and the alkyl moiety in the alkylthio group or thealkoxy group may also be either a straight chain or a branched alkylgroup. The alkyl group having an alkylthio group is preferably analkylthiomethyl group or an alkylthioethyl group, and the alkylthiogroup is preferably the one containing 1 or 2 carbon atoms. Thepreferred are methylthiomethyl group, ethylthiomethyl group, andmethylthioethyl group. The alkyl group having an alkoxy group ispreferably an alkoxymethyl group or an alkoxyethyl group, and the alkoxygroup is preferably the one containing 1 or 2 carbon atoms. Thepreferred are methoxymethyl group, ethoxymethyl group, and methoxyethylgroup. The more preferred are methylthiomethyl group and methoxymethylgroup.

When R³ is a cycloalkyl group, the substituent is one or more groupselected from the group consisting of an alkyl group containing 1 to 6carbon atoms, halogen atom, amino group, and hydroxy group. Preferableexamples of such substituent are methyl group, ethyl group, fluorineatom, and chlorine atom.

Preferable examples of R³ include those containing 1 or 2 carbon atomssuch as methyl group; ethyl group; vinyl group; fluoro-substitutedmethyl group or ethyl group; methyl group or ethyl group having aminogroup or hydroxy group; and methyl group having thiomethyl group ormethoxy group. R³ is most preferably methyl group or ethyl group.

R⁴ and R⁵ independently represent hydrogen atom, halogen atom, an alkylgroup containing 1 to 6 carbon atoms, an alkoxy group containing 1 to 6carbon atoms, an alkenyl group containing 2 to 6 carbon atoms, analkynyl group containing 2 to 6 carbon atoms, or an optionallysubstituted cycloalkyl group containing 3 to 6 carbon atoms. When R⁴ orR⁵ is an alkyl group, it may be substituted with a substituent selectedfrom the group consisting of hydroxy group, amino group, halogen atom,an alkylthio group containing 1 to 6 carbon atoms, and an alkoxy groupcontaining 1 to 6 carbon atoms. R⁴ and R⁵ are not simultaneouslyhydrogen atom.

R⁴ and R⁵ may also combine together to form

(a) a 3- to 6-membered cyclic structure including the carbon atom sharedby R⁴ and R⁵ to form a spirocyclic structure with the pyrrolidine ring,the thus formed spiro ring optionally containing oxygen atom or sulfuratom as a ring member atom, and optionally being substituted with ahalogen atom or an alkyl group containing 1 to 6 carbon atoms optionallyhaving a substituent; or

(b) exomethylene group bonding to the pyrrolidine ring by double bond,the exomethylene group optionally having 1 or 2 substituents selectedfrom hydroxy group, amino group, halogen atom, an alkylthio groupcontaining 1 to 6 carbon atoms, and an alkoxy group containing 1 to 6carbon atoms.

When R⁴ or R⁵ is an alkyl group, it may be either a straight chain or abranched alkyl group, and it may be methyl group, ethyl group, propylgroup, or isopropyl group; more preferably methyl group or ethyl group;and most preferably methyl group.

When R⁴ or R⁵ is an alkyl group and this alkyl has hydroxy group oramino group as its substituent, the alkyl group is preferablysubstituted with such substituent at its terminal carbon atom. The alkylgroup having hydroxy group is preferably the one containing up to 3carbon atoms, and preferable examples include hydroxymethyl group,2-hydroxyethyl group, 2-hydroxypropyl group, and 3-hydroxypropyl group.The alkyl group having amino group is preferably the one containing upto 3 carbon atoms, and preferable examples include aminomethyl group,2-aminoethyl group, 2-aminopropyl group, and 3-aminopropyl group.

When R⁴ or R⁵ is an alkyl group and this alkyl group has a halogen atomas its substituent, the alkyl group may be either a straight chain or abranched alkyl group containing 1 to 6 carbon atoms, and the halogenatom is preferably fluorine atom. The number of the fluorinesubstitution is not limited and the substitution may be mono- toperfluoro substitution. Exemplarily preferable substituents ashalogenated alkyl group are halogen-substituted alkyl groups includemonofluoromethyl group, difluoromethyl group, trifluoromethyl group, and2,2,2-trifluoroethyl group.

When R⁴ or R⁵ is an alkyl group having an alkylthio group or an alkoxygroup as its substituent, the alkyl group may be either a straight chainor a branched alkyl group, and the alkyl moiety in the alkylthio groupor the alkoxy group may also be a straight chain or a branched alkylgroup. The alkyl group having an alkylthio group is preferably analkylthiomethyl group, an alkylthioethyl group, or an alkylthiopropylgroup, and the alkylthio group is preferably the one containing 1 to 3carbon atoms. More preferably, the alkyl group having an alkylthio groupis methylthiomethyl group, ethylthiomethyl group, or methylthioethylgroup. The alkyl group having an alkoxy group is preferably analkoxymethyl group, an alkoxyethyl group, or an alkoxypropyl group, andthe alkoxy group is preferably the one containing 1 to 3 carbon atoms.More preferably, the alkyl group having an alkoxy group is methoxymethylgroup, ethoxymethyl group, or methoxyethyl group.

When R⁴ or R⁵ is a cycloalkyl group, it is preferably cyclopropyl groupor cyclobutyl group and more preferably cyclopropyl group. When R⁴ or R⁵is a substituted cycloalkyl group, the substituent may be the same asthe same case of R³, and is at least one substituent selected from thegroup consisting of an alkyl group containing 1 to 6 carbon atoms,halogen atom, amino group, and hydroxy group. Preferable examples ofsuch substituent are methyl group, ethyl group, fluorine atom, andchlorine atom.

When R⁴ or R⁵ is a halogen atom, it may be fluorine atom, chlorine atom,or iodine atom, and preferably, fluorine atom.

When R⁴ or R⁵ is an alkoxy group, it may be any of the alkoxy groupsderived from the alkyl group as described above, and it is preferably analkoxy group containing 1 to 3 carbon atoms. Exemplary such alkoxygroups include methoxy group and ethoxy group.

When R⁴ or R⁵ is an alkenyl group or an alkynyl group, these groups maybe as defined above for R³.

When R⁴ and R⁵ combine together to form a spirocyclic structure, R⁴ andR⁵ together form a polymethylene chain containing 2 to 5 carbon atomsand opposite ends of the thus formed polymethylene chain bind to thecarbon atom having the R⁴ and R⁵ attached thereto to thereby for acyclic structure. The thus formed ring may have a size of 3-memberedring to six-membered ring, and among these, the preferred are 3-memberedring or 4-membered ring, and the more preferred are 3-membered ring. Themethylene group in the polymethylene chain may be replaced with oxygenatom or sulfur atom to form a saturated heterocycle. The ring formed byR⁴ and R⁵ is optionally substituted with a halogen atom or an optionallysubstituted alkyl group containing 1 to 6 carbon atoms. Exemplaryhalogen atoms include fluorine atom and chlorine atom. The alkyl groupmay be either a straight chain or a branched alkyl group, and thepreferred are methyl group, ethyl group, propyl group, and isopropylgroup, and the more preferred are methyl group or ethyl group. Thisalkyl group is optionally substituted with a substituent which ispreferably a halogen atom.

When R⁴ and R⁵ together form an exomethylene group which binds to thepyrrolidine ring by double bond, a carbon-carbon double bond is formedby using the carbon atom at position 4 of the pyrrolidinyl group havingthe R⁴ and R⁵ attached thereto as one of the carbon atom. In such acase, the pyrrolidinyl substituent moiety has a structure represented bythe following formula:

wherein R⁴¹ and R⁵¹, both represents hydrogen atoms, or one of themrepresents a hydrogen atom and the other represents a substituentselected from the group consisting of hydroxy group, amino group,halogen atom, an alkylthio group containing 1 to 6 carbon atoms, or analkoxy group containing 1 to 6 carbon atoms.

When the substituent of the exomethylene group is alkylthio group oralkoxy group, the alkyl moiety thereof may be optionally substitutedwith a substituent selected from the group consisting of hydroxy group,amino group, halogen atom, an alkylthio group containing 1 to 6 carbonatoms, and an alkoxy group containing 1 to 6 carbon atoms. Among these,the an alkylthio group containing 1 to 6 carbon atoms or the an alkoxylgroup containing 1 to 6 carbon atoms is preferably an alkylthio oralkoxy group containing 1 to 3 carbon atoms, more preferably, methylthiogroup, ethylthio group, methoxy group, or ethoxy grout, and still morepreferably methylthio group or methoxy group.

Preferably, the exomethylene group is not substituted with a substituentother than hydrogen atom. However when the exomethylene group has asubstituent, the substituent is preferably hydroxy group, amino group,fluorine atom, chlorine atom, methylthio group, or methoxy group.

Preferable combination of R⁴ and R⁵ is the one wherein one of R⁴ and R⁵is hydrogen atom and the other is fluorine atom, methyl group, ethylgroup, normal-propyl group, isopropyl group, normal-butyl group,cyclopropyl group, fluoromethyl group, methoxy group, vinyl group, orethynyl group. Also preferred are R⁴ and R⁵ together formingcyclopropane ring or cyclobutane ring including the carbon atom sharedby R⁴ and R⁵ to form a spirocyclic structure. Further, R⁴ and R⁵preferably combine together forming an exoalkylene group containing 2 to5 carbon atoms.

R⁴ or R⁵ is preferably a fluoroalkyl group, a fluoline atom, orspirocyclic structure or exomethylene group by the constructed by thecombination of these

R⁶ and R⁷ independently represent hydrogen atom or an alkyl groupcontaining 1 to 6 carbon atoms. The alkyl group may be either a straightchain or a branched alkyl group, and the preferred are methyl group,ethyl group, propyl group, and isopropyl group. The more preferred aremethyl group or ethyl group, and the most preferred is methyl group.Preferably, both R⁶ and R⁷ are hydrogen atom.

R⁸ represents a halogen-substituted alkyl group containing 1 to 6 carbonatoms, a halogen-substituted cycloalkyl group containing 3 to 6 carbonatoms, a halogen-substituted phenyl group, or a halogen-substitutedheteroaryl group.

When R⁸ is a halogen-substituted alkyl group containing 1 to 6 carbonatoms, the alkyl group moiety may be either a straight chain or abranched group as exemplified by methyl group, ethyl group, propylgroup, isopropyl group, butyl group, isobutyl group, sec-butyl group,and tert-butyl group. Among these, the preferred is ethyl group. Thehalogen atom substituting the alkyl group is preferably fluorine atom orchlorine atom, and more preferably fluorine atom. Examples of thehalogen-substituted alkyl group include fluoromethyl group,1-fluoroethyl group, and 2-fluoroethyl group, and the preferred is2-fluoroethyl group.

When R⁸ is a halogen-substituted cycloalkyl group containing 3 to 6carbon atoms, exemplary cyclic alkyl group include cyclopropyl group,cyclobutyl group, and cyclopentyl group, and the preferred iscyclopropyl group. Exemplary substituent halogen atoms include fluorineatom and chlorine atom, and the preferred is fluorine atom.Mono-substitution with the halogen atom is sufficient, and the preferredis monofluorocyclopropyl group, and the more preferred iscis-monofluorocyclopropyl group.

The halogen atom in the halogen-substituted phenyl group is preferablyfluorine atom or chlorine atom, and more preferably, fluorine atom. Thesubstitution with the halogen atom is preferably a mono- ordi-substitution. The halogen-substituted phenyl groups is preferably2-fluorophenyl group, 4-fluorophenyl group, or 2,4-difluorophenyl group.

The heteroaryl group in the halogen-substituted heteroaryl group may bea five-membered or a six-membered aromatic heterocyclic group containingone or more heteroatoms selected from nitrogen atom, sulfur atom, andoxygen atom. Among such heteroaryl groups, the preferred is afive-membered or a six-membered nitrogen-containing aromaticheterocyclic group containing 1 or 2 nitrogen atoms. Exemplary suchgroups include pyridyl group, pyrimidyl group, piperidinyl group,pyrrolidinyl group, morpholinyl group, pyrrolyl group, imidazolyl group,pyrazolyl group, pyridyl group, pyrimidinyl group, pyridazinyl group,pyrrolidinyl group, pyrrolynyl group, imidazolidinyl group, imidazolinylgroup, pyrazolidinyl group, pyrazolinyl group, piperidyl group, andpiperazinyl group, and among these, the preferred is pyridyl group. Thehalogen atom is preferably fluorine atom or chlorine atom, and morepreferably fluorine atom. The substitution with the halogen atom ispreferably a mono- or di-substitution.

R⁸ is preferably a halogen-substituted cycloalkyl group containing 3 to6 carbon atoms, and preferably, a 2-halogenocyclopropyl group, and morepreferably a 1,2-cis-2-halogenopropyl group, and particularly, a(1R,2S)-2-halogenocyclopropyl group. The more preferable ismonofluorocyclopropyl group, and in particular,cis-monofluorocyclopropyl group. The most preferable is1,2-cis-2-fluorocyclopropyl group, and in particular,(1R,2S)-2-fluorocyclopropyl group.

R⁹ represents hydrogen atom, phenyl group, acetoxymethyl group, pivaloyloxymethyl group, ethoxycarbonyl group, choline group, dimethylaminoethyl group, 5-indanyl group, phthalidinyl group,5-alkyl-2-oxo-1,3-dioxol-4-ylmethyl group, 3-acetoxy-2-oxobutyl group,an alkyl group containing 1 to 6 carbon atoms, an alkoxymethyl groupcontaining 2 to 7 carbon atoms, or a phenylalkyl group comprising analkylene group containing 1 to 6 carbon atoms and phenyl group.

R⁹ is preferably hydrogen atom.

X¹ represents hydrogen atom or a halogen atom. Preferable halogen atomis fluorine atom or chlorine atom, and the more preferred is fluorineatom. X¹ is preferably fluorine atom or hydrogen atom.

A represents nitrogen atom or a moiety represented by formula (II):

wherein X² represents an alkyl group containing 1 to 6 carbon atoms, analkoxy group containing 1 to 6 carbon atoms, hydrogen atom, cyano group,halogen atom, a halogen-substituted methyl group, or a halogenomethoxygroup. X² together with R⁸ may combine together to form a cyclicstructure including a part of the mother nucleus, and the thus formedring may optionally contain oxygen atom, nitrogen atom, or sulfur atomas a ring member atom, and the ring may be substituted with an alkylgroup containing 1 to 6 carbon atoms optionally having a substituent.

When A is a moiety represented by formula (II), and X² is an alkyl groupcontaining 1 to 6 carbon atoms, X² may be either a straight chain or abranched alkyl group. Preferably, X² is methyl group, ethyl group,propyl group, or isopropyl group, and among these, the preferred aremethyl group and ethyl group, and the more preferred is methyl group.When X² is an alkoxy group containing 1 to 6 carbon atoms, it may be anyof the alkoxy group derived from the alkyl group as described above.Among those described above, X² is preferably an alkyl group containing1 to 3 carbon atoms or an alkoxy group containing 1 to 3 carbon atoms,and more preferably methyl group or methoxy group.

When X² is a halogen atom, it is preferably fluorine atom or chlorineatom, and more preferably fluorine atom. When X² is ahalogen-substituted methyl group, the halogen atom is preferablyfluorine atom or chlorine atom, and more preferably fluorine atom.Exemplary halogen-substituted methyl groups include fluoromethyl group,difluoromethyl group, and trifluoromethyl group. Similarly, when X² is ahalogenomethoxy group, the halogen atom is preferably fluorine atom orchlorine atom, and more preferably, fluorine atom. Exemplaryhalogenomethoxy groups include fluoromethoxy group, difluoromethoxygroup, and trifluoromethoxy group.

When A is a moiety represented by formula (II), X² and R⁸ may combinetogether to form a cyclic structure containing a part of the quinoloneskeleton (3 atoms, namely, the carbon atom having X² bonded thereto; thenitrogen atom having R⁸ bonded thereto; and the carbon atom between thenuclei having X² and R⁸ bonded thereto). The ring formed may preferablyhave a size of 5 to 7-membered ring, and the ring may be eithersaturated or unsaturated. This cyclic structure may also contain oxygenatom, nitrogen atom, or sulfur atom as a constituent atom of the ring,and this cyclic structure may be further substituted with an alkyl groupcontaining 1 to 6 carbon atoms as described above for X². The cyclicstructure preferably contains oxygen atom, and it is preferablysubstituted with a methyl group. Such partial structure is preferably astructure represented by the formula: —O—CH₂—CH(—CH₃)— (the carbon atomat the right end binds to nitrogen atom).

When A is a moiety represented by formula (II), and the substituent X²does not form a cyclic structure, X² is preferably methyl group, ethylgroup, methoxy group, difluoromethoxy group, cyano group, or chlorineatom, and most preferably methyl group, methoxy group, ordifluoromethoxy group.

When A is a moiety represented by formula (II), and the substituent X²forms a cyclic structure, the preferred is the formation of2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid skeleton, and in particular, 3-(S)-methylpyridobenzoxazineskeleton.

The compound of the present invention has a characteristic feature thatit has a substituent represented by the following formula:

at position 7 (or at an equivalent position) of the quinoline skeleton.

In other words, compound of the present invention has a characteristicfeature that amino group is present at position 3 of the pyrrolidinylgroup; the carbon atom having this amino group bonded thereto has asubstituent R³ which is not hydrogen atom; and the carbon atom atposition 4 is mono- or di-substituted. That is, position 3 of the1-pyrrolidinyl group is di-substituted by a substituent including the3-amino group; and position 4 is mono- or di-substituted; andaccordingly, positions 3 and 4 are tri- or tetra-substituted.

This pyrrolidinyl group contains an asymmetric carbon atom, andaccordingly, stereoisomers are present as described below. First, twostereoisomers:

are present for the position 3.

When both R⁴ and R⁵ are not hydrogen atom (including the case when R⁴and R⁵ together form a structure), the following structure:

wherein the amino group is at β-configuration is preferred.

When either one of R⁴ and R⁵ is hydrogen atom, the following 4 types:

are present. The one having structure 1 is generally more preferablethan the one having structure 4, while the actual preferable formchanges according to the structure of the substituent R⁵. All of thestructures are within the scope of the present invention.

Preferable basic skeletons for the quinolone carboxylic acid having asubstituent at position 7 are as shown below.

Preferable substituents at position 7 are as shown below.

Accordingly, the preferable compound of the present invention is thequinolone carboxylic acid basic skeleton as mentioned above substitutedwith the substituent at position 7 as mentioned above (namely, thecombination of the basic skeleton core and the substituent as mentionedabove). In the formulae as shown above, absolute configuration atposition 3 of the pyrrolidine ring substituted with the amino group iseither 3R or 3S. Preferably, the compound of the present invention isstereochemically pure.

Preferred examples of the compound of the present invention is asdescribed below.

-   7-[3-amino-3,4-dimethylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-3,4-dimethylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-3,4-dimethylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-ethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-ethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-4-ethyl-3-methylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-3-methyl-4-isopropyl    pyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-3-methyl-4-isopropyl    pyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-3-methyl-4-isopropyl    pyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-cyclopropyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-cyclopropyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-4-cyclopropyl-3-methylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-3-methyl-4-vinylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-3-methyl-4-vinylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-3-methyl-4-vinylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-methylene-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-methylene-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-4-methylene-3-methylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3R)-3-amino-3-methyl-4-methylene    pyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-(3-amino-4-methoxy-3-methylpyrrolidine-1-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-(3-amino-4-methoxy-3-methylpyrrolidine-1-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S,4S)-3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S,4S)-3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S,4S)-3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S,4S)-3-amino-4-fluoromethyl-3-methylpyrrolidine-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3R)-3-amino-4-fluoro-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3R)-3-amino-4-fluoro-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S)-3-amino-3-fluoromethyl-4-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(3S)-3-amino-3-fluoromethyl-4-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-9-benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[8-amino-8-methyl-6-azaspiro[3.4]octane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methyl-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[8-amino-8-methyl-6-azaspiro[3.4]octane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-1,4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   (3S)-10-[8-amino-8-methyl-6-azaspiro[3.4]octane-5-yl]-9fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]-benzoxazine-6-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-1-cyclopropyl-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof,-   7-[(7S)-7-amino-7-methoxy-5-azaspiro[2.4]heptane-5-yl]-1-cyclopropyl-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylic    acid, or a salt or a hydrate thereof.

Next, method for synthesizing 3-amino-3-aliphatichydrocarbon-substituted-4-aliphatic hydrocarbon-substituted pyrrolidinederivative which is relevant with the present invention is described.Typical example of the 3-amino-3-aliphatichydrocarbon-substituted-4-aliphatic hydrocarbon-substituted pyrrolidinederivative is 3-amino-3-aliphatic hydrocarbon-substituted-4-aliphatichydrocarbon-substituted pyrrolidine-1-yl group such as3-amino-3-methyl-4-alkyl-substituted pyrrolidine-1-yl group.

3-Amino-3-methyl-4-alkyl-substituted pyrrolidine derivative (8) which isa typical substituent compound in the present invention can be producedby synthesizing an important intermediate by 1,3-dipolar cycloadditionusing 3-substituted crotonate ester (1) and azomethine ylide (2) for thereaction block, followed by hydrolysis of the ester moiety andconversion into amine. Although the inventors of the present inventionselected tertiary butoxycarbonyl group for the protective group of theamine moiety at position 3, the protective group of the amine moiety atposition 3 is not limited to the tertiary butoxycarbonyl group as longas the selected protective group does not affect the subsequent reactionsteps and is easily deprotected later, and a protective group which isthe same as the protective group at position 1 may also be used.Synthesis of the optically active compound can be conducted, forexample, by optical resolution using an appropriate intermediate, forexample, by HPLC resolution using a chiral column of the appropriateintermediate, preferential crystallization of the diastereomer salt, orby bonding chiral building block to an appropriate intermediate toproduce a diastereomer, separating the diastereomer by using anappropriate separation technique such as silica gel chromatography, andremoving the chiral building block to produce an optically activesubstance. Alternatively, the optically active compound may besynthesized by using the chiral building block for the startingmaterial.

In the formula, Boc represents tertiary butoxycarbonyl group, Cbzrepresents benzyloxycarbonyl group, R¹⁰ represents an alkyl groupcontaining 1 to 6 carbon atoms, and R¹¹ represents R⁴ or R⁵ describedfor the formula (I) excluding hydrogen atom.

Step 1 is a step wherein 3-alkoxycarbonyl-3-methyl-4-substitutedpyrrolidine derivative (3) is synthesized by 1,3-dipolar cycloadditionby using 3-substituted crotonate ester (1) and azomethine ylide (2) forthe reaction blocks. The azomethine ylide used for producing theazomethine ylide may be produced, for example, by adding a catalyticamount of trifluoroacetic acid or a catalytic amount of silver fluorideto N-benzyl-N-(methoxymethyl) trimethylsilyl methylamine (See Journal ofOrganic Chemistry, vol. 52, No. 2, page 235, 1987). The reaction solventis not particularly limited as long as it produces azomethine ylidewithout inhibiting the 1,3-dipolar cycloaddition. The solvent, however,is preferably dichloromethane or 1,2-dichloroethane. The reaction may beconducted at a temperature of from −20° C. to the reflux temperature ofthe solvent, and preferably, at room temperature to the refluxtemperature of the solvent.

Step 2 is the step in which protective group at position 1 of thepyrrolidine ring is converted. This step is preferably conducted inorder to enable separation and purification by extraction of thecarboxylic acid derivative produced by the hydrolysis of the ester atposition 3. The protective group at position 1 is preferably the onewhich is distinguishable in the deprotection step from the protectivegroup of the amino group at position 3 generated in the subsequentconversion although use of the same protective group is allowable.Preferable protective group at position 1 is benzyloxycarbonyl group.The reaction for introducing this benzyloxycarbonyl is generallyconducted by direct conversion by von Braun reaction using benzylchloroformate in a solvent such as dichloromethane; by catalytichydrolysis using a catalyst such as palladium-carbon followed byreaction with benzyl chloroformate in an appropriate solvent and in thepresence of a base.

Step 3 is the step of hydrolyzing the ester at position 3 of thepyrrolidine ring. The ester is an alkyl ester containing 1 to 6 carbonatoms, and preferably, methyl ester, ethyl ester, or tertiary butylester. The hydrolysis is can be conducted by any method commonly used inthe art as long as the protective group at position 1 is not affected,and typically by hydrolysis using a base or an acid. Hydrolysis of themethyl ester and the ethyl ester is conducted by reaction with arealkaline aqueous solution such as aqueous solution of sodium hydroxide,aqueous solution of potassium hydroxide, or aqueous solution of bariumhydroxide in ethanol or water followed by acidification by anappropriate acid which does not affect the protective group at position1 for separation and purification. In the case of hydrolysis of thetertiary butyl ester, the hydrolysis is conducted under acidicconditions or in the presence of an acid catalyst in an appropriatesolvent in which the ester is soluble. Preferable acids includehydrochloric acid, formic acid, acetic acid, trifluoroacetic acid, andtoluenesulfonic acid.

Step 4 is the step of converting the carboxyl group at position 3 of thepyrrolidine ring to amino group. This step is generally carried out byrearrangement of carboxylic acid to amine. For example, when therearrangement is accomplished by Curtius rearrangement, the carboxylicacid is converted to acid azide by using a reagent such as sodium azide,trimethylsilyl azide, or diphenylphosphoryl azide (DPPA) in anappropriate solvent such as toluene, and converting the acid azide toisocyanate by heating the reaction mixture, and then converting theisocyanate to amine by hydrolysis using hydrochloric acid or the like.

Step 5 is the step of protecting the amino group at position 3 of thepyrrolidine ring. However, the subsequent steps may also be conductedwithout protecting the amino group. The protective group of the aminogroup at position 3 may be an amino protecting group commonly used inthe art. However, use of a protective group which is distinguishable inthe deprotection step from the protective group at position 1 ispreferable. Examples include tertiary butoxycarbonyl group, acetylgroup, and trifluoroacetyl group, and the preferred is tertiarybutoxycarbonyl group.

It should be noted that Step 4 and Step 5 can be accomplished in onestep when the rearrangement is conducted in an appropriate solvent. Forexample, Curtis rearrangement may be carried out by usingdiphenylphosphoryl azide (DPPA) in tertiary butyl alcohol to produce3-(tertiary butoxycarbonyl)aminopyrrolidine derivative.

Step 6 is the step of deprotection of position 1 of the pyrrolidinering, and the deprotection reaction may be conducted under anyconditions as long as other functional groups and configuration are notaffected. Since the protective group at position 1 is benzyloxycarbonylgroup with regard to the compound of the present invention, thedecomposition is conducted under deprotection conditions commonly usedin the art, for example, by catalytic hydrolysis in the presence of acatalyst such as palladium-carbon or by using ammonium formate in aprotic polar solvent. When carbon-carbon unsaturated bond is present atposition 4 of the pyrrolidine ring due to the presence of thesubstituent such as vinyl group or methylene group, the decompositionshould be accomplished while maintaining the carbon-carbon unsaturatedbond. Since the protective group at position 1 is benzyloxycarbonylgroup with regard to the compound of the present invention, thedecomposition condition capable of maintaining the carbon-carbonunsaturated bond of the vinyl group, methylene group, or the like atposition 4 of the pyrrolidine ring is provided by the method usingsodium-liquid ammonia (Birch reduction conditions) in the presence of astrong acid (for example, hydrobromic acid-acetic acid, trifluoroaceticacid, and trifluoromethanesulfonic acid-trifluoroacetic acid), themethod using barium hydroxide, and the like.

Next, synthesis of pyrrolidine derivative which is typically7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl group is described.

7-amino-7-methyl-5-azaspiro[2.4]-heptane derivative (17) which isanother typical compound of the present invention can be synthesized byconverting ketone moiety of acetoacetate derivative into aminonitrilederivative by Strecker reaction, converting cyano group to aminomethylgroup by reduction, and condensing the aminomethyl group with estermoiety (carboxylic acid unit) to produce a pyrrolidone derivative whichis an important intermediate.

Although the inventors of the present invention selected tertiarybutoxycarbonyl group for the protective group of the amine moiety atposition 3, the protective group of the amine moiety at position 3 isnot limited to the tertiary butoxycarbonyl group as long as the selectedprotective group does not affect the subsequent reaction steps and iseasily deprotected later, and a protective group which is the same asthe protective group at position 1 may also be used. Synthesis of theoptically active compound can be conducted, for example, by opticalresolution using an appropriate intermediate, for example, by HPLCresolution using a chiral column of the appropriate intermediate,preferential crystallization of the diastereomer salt, or by bondingchiral building block to an appropriate intermediate to produce adiastereomer, separating the diastereomer by using an appropriateseparation technique such as silica gel chromatography, and removing thechiral building block to produce an optically active substance.Alternatively, the optically active compound may be synthesized by usingthe chiral building block for the starting material.

In the formula, Boc represents tertiary butoxycarbonyl group, and R¹²represents an alkyl group containing 1 to 6 carbon atoms.

Step 7 is the step of constructing a cyclic structure at methylenemoiety of the acetoacetate derivative. This step can be generallyaccomplished by using a 1,2-dihalogeno ethane such as dibromoethane asan alkylating agent in the presence of a base. Exemplary bases includepotassium carbonate, sodium hydride, and metal sodium, and the exemplaryreaction solvents include acetone and N,N-dimethylformamide. Aftercompleting the reaction, the cyclo compound may be separated andpurified by distillation under a reduced pressure.

Step 8 is the step of converting methylketone moiety to aminonitrilederivative by Strecker reaction. This Strecker reaction is conducted byreacting ammonia with a cyanating agent such as potassium cyanideoptionally in the presence of ammonium chloride. The reaction conditionsmay be adequately selected by referring to the Strecker reactioncommonly used in the amino acid synthesis.

Step 9 is the step of reducing cyano group for conversion intomethylamine. The reduction of nitrile can be generally accomplished bycatalytic reduction in the presence of a catalyst in an appropriatesolvent such as ethanol. Examples of the catalyst includepalladium-carbon catalyst, Raney nickel, Raney cobalt, and platinumoxide. When secondary amine is produced as a byproduct in the catalyticreduction of nitrile, the reduction may be conducted in the presence ofammonia. The reduction may be carried out by a metal hydride if otherfunctional groups in the reaction system, for example, ester group whichis the typical example in the compound of the present invention is notreduced. A typical example of the metal hydride is sodiumborohydride-cobalt chloride (II). If the ester moiety is reduced, thereaction may be conducted after converting the ester moiety to a bulkyester such as tertiary butyl ester which is not reduced.

Step 10 is the step of condensing ester moiety (carboxylic acid unit)and methylamine in the molecule to produce the pyrrolidone derivative.When the ester moiety is methyl ester or ethyl ester, the condensationcan be generally accomplished by heating the reaction solution from theroom temperature in an appropriated solvent. When the ester moiety ismethyl ester or ethyl ester, the pyrrolidone derivative can be directlyproduced from the reaction of Step 9. On the other hand, when the esteris a bulky ester such as tertiary butyl ester, the condensation isaccomplished by hydrolyzing the ester by a method commonly used in theart, and then converting the hydrolyzate into the pyrrolidone derivativeby using a condensing agent such as DCC.

Step 11 is the step of protecting the amino group at position 3 of thepyrrolidine ring. However, the subsequent steps may also be conductedwithout protecting the amino group. The protective group of the aminogroup at position 3 may be an amino protecting group commonly used inthe art which is stable under the reaction conditions of the subsequentstep 13. However, use of a protective group which is distinguishable inthe deprotection step from the protective group at position 1 ispreferable. Examples include tertiary butoxycarbonyl group, acetylgroup, and trifluoroacetyl group, and the preferred is tertiarybutoxycarbonyl group.

Step 12 is the step of protecting position 1 of the pyrrolidine ring.However, the subsequent steps may also be conducted without protectingthe amino group. The protective group of position 1 may be an aminoprotecting group commonly used in the art which is stable under thereaction conditions of the subsequent step 13. However, use of aprotective group which is distinguishable in the deprotection step fromthe amino group protective group at position 3 is preferable. Theinventors of the present invention selected benzyl group for theprotective group. The reaction of introducing the benzyl group isconducted by using benzyl halide in the presence of a base such assodium hydride or potassium carbonate. The reaction solvent used may beacetone, N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof.

Step 13 is the step of reducing carbonyl group of the pyrrolidone. Thereduction is conducted by using a reducing agent. Exemplary reducingagents include metal hydrides such as lithium aluminum hydride, andsodium bis(2-methoxyethoxy) aluminum hydride, boron hydride compoundssuch as diborane and borane-tetrahydrofuran complex. The solvent used istypically ether solvent such as tetrahydrofuran, and the reaction may becarried out at a temperature of −78° C. to 100°.

Step 14 is the step or deprotecting position 1 of the pyrrolidine ring,and the deprotection reaction may be conducted under any conditions aslong as other functional groups and configuration are not affected.Since the protective group at position 1 is benzyl group with regard tothe compound of the present invention, the decomposition is conductedunder deprotection conditions commonly used in the art, for example, bycatalytic hydrolysis in the presence of a catalyst such aspalladium-carbon or by using ammonium formate in a protic polar solvent.When carbon to carbon unsaturated bond is present at position 4 of thepyrrolidine ring due to the presence of the substituent such as vinylgroup or methylene group, the decomposition should be accomplished whilemaintaining the carbon to carbon unsaturated bond. Since the protectivegroup at position 1 is benzyl group with regard to the compound of thepresent invention, the decomposition condition capable of maintainingthe carbon to carbon unsaturated bond of the vinyl group, methylenegroup, or the like at position 4 of the pyrrolidine ring is provided,for example, by the method using sodium-liquid ammonia (Birch reductionconditions).

In the foregoing, the reactions have been described in terms ofexamples. Those skilled in the art will be able to find a new syntheticmethod by taking such reactions into consideration. The scope of thepresent invention is not limited by the reactions as described above.

When the compound of the present invention (I) is produced by using thethus produced Compound (8) or Compound (17), a compound having thequinolone skeleton represented by the following formula (18):

wherein R⁸, X¹, and A are as defined above; and R⁹¹ represents hydrogenatom, dihalogenoboron, or diacyloxy boron; and X² represents a leavinggroup may be reacted with Compound (8) or Compound (17).

R⁹¹ of the compound having the quinolone skeleton is hydrogen atom or aboron substituent capable of producing a boron chelate. The boronsubstituents may be a dihalogenoboron or a diacyloxy boron. Preferabledihalogenoboron is difluoroboron (—BF₂), and preferable diacyloxy boronis diacetyloxy boron [—B(OAc)₂], and these compound can be produced bythe method commonly used in the art.

The production method is described by using the compound of Example 9 asan example.

The target compound can be produced by dissolving the compound havingthe quinolone skeleton in an appropriate solvent and reacting thecompound having the quinolone skeleton with Compound (8) or (17) forintroducing the substituent at position 7 in the presence of a base. Theamino group of the compound for introducing the substituent at position7 may be protected with a protective group, and exemplary protectivegroups other than tert-butyl oxycarbonyl (Boc) include benzyloxycarbonylgroup, p-methoxy benzyloxycarbonyl group, acetyl group, methoxyacetylgroup, trifluoroacetyl group, pivaloyl group, formyl group, benzoylgroup, tert-butyl group, benzyl group, trimethylsilyl group, andisopropyl dimethylsilyl. Exemplary bases include carbonate, hydrogencarbonate, or hydroxide of an alkaline metal or an alkaline earth metal;a trialkylamine such as triethylamine and N,N-diisopropylethylamine; andnitrogen-containing heterocyclic group compounds such as pyridine,1,8-diazabicycloundecene, and N-methylpiperidine, and the preferred aretrialkylamines, and in particular triethylamine. The solvent used is notparticularly limited as long as it does not inhibit the reaction, andpreferable examples include N,N-dimethylformamide, dimethyl sulfoxide,sulfolane, acetonitrile, ethanol, dimethyl acetamide, tetrahydrofuran,and N-methylpyrrolidone, and the more preferred are dimethyl sulfoxideor sulfolane.

When the compound having the quinolone skeleton is a boron chelatecompound, the target compound can be produced by cleaving the boronsubstituent moiety by hydrolysis, and deprotecting the protective groupof the amino group. The hydrolysis of the boron substituent can beconducted under conditions commonly used in the art, for example, byreacting with a base in an alcohol solvent such as methanol and ethanol.The base is preferably triethylamine, and the reaction is preferablyconducted in an ice bath. The deprotection is conducted under theconditions suitable for the protective group used, for example, bytreating the hydrolyzate with concentrated hydrochloric acid. After thereaction, the reaction solution is alkalized by adding an aqueoussolution of sodium hydroxide.

Accordingly, the compounds represented by the following formulae (19)and (20) are useful as an intermediate for producing the compound (I) ofthe present invention.

In the formulae, R¹¹ represents a group comprising R¹ as defined above(hydrogen atom, an alkyl group containing 1 to 6 carbon atoms, acycloalkyl group containing 3 to 6 carbon atoms, or a substitutedcarbonyl group derived from an amino acid, a dipeptide, or a tripeptide;the alkyl group being optionally substituted with a substituent selectedfrom the group consisting of hydroxy group, amino group, halogen atom,an alkylthio group containing 1 to 6 carbon atoms, and an alkoxy groupcontaining 1 to 6 carbon atoms) and the protective group of the aminoacid;

R²¹ represents a group comprising R² as defined above (hydrogen atom, analkyl group containing 1 to 6 carbon atoms, or a cycloalkyl groupcontaining 3 to 6 carbon atoms; the alkyl group being optionallysubstituted with a substituent selected from the group consisting ofhydroxy group, amino group, halogen atom, an alkylthio group containing1 to 6 carbon atoms, and an alkoxy group containing 1 to 6 carbon atoms)and the protective group of the amino acid; and

R³, R⁴, R⁵, R⁶, and R⁷ are as defined above.

Next, the protective group of the amino group represented by R¹¹ and R²¹is described. The protective group is not particularly limited as longthe protective group is the one widely used in the art, and exemplaryprotective groups include alkoxycarbonyl groups such as tartiarybutlxycarbonyl group, and 2,2,2-trichloroethoxycarbonyl group;aralkyloxycarbonyl groups such as benzyloxycarbonyl group,paramethoxybenzyloxycarbonyl group, and paranitrobenzyloxycarbonylgroup; acyl groups such as acetyl group, methoxyacetyl group,trifluoroacetyl group, chloroacetyl group, pivaloyl group, formyl group,and benzoyl group; alkyl groups or aralkyl groups such as tertiary butylgroup, benzyl group, paranitrobenzyl group, paramethoxybenzyl group, andtriphenylmethyl group; ethers such as methoxymethyl group, tertiarybutoxymethyl group, tetrahydropyranyl group, and2,2,2-trichloroethoxymethyl group; (alkyl and/or aralkyl)-substitutedsilyl groups such as trimethylsilyl group, isopropyldimethylsilyl group,tertiary butyldimethylsilyl group, tribenzylsilyl group, and tertiarybutyldiphenylsilyl group.

In the formula R¹³ represents protective group of the amino group, andR¹¹, R²¹, R³, R⁴, R⁵, R⁶ and R⁷ are as defined above.

The protective group represented by R¹³ is not particularly limited aslong the protective group is the one widely used in the art. Exemplaryprotective groups include alkoxycarbonyl groups such as tartiarybutlxycarbonyl group, and 2,2,2-trichloroethoxycarbonyl group;aralkyloxycarbonyl groups such as benzyloxycarbonyl group,paramethoxybenzyloxycarbonyl group, and paranitrobenzyloxycarbonylgroup; acyl groups such as acetyl group, methoxyacetyl group,trifluoroacetyl group, chloroacetyl group, pivaloyl group, formyl group,and benzoyl group; alkyl groups or aralkyl groups such as tertiary butylgroup, benzyl group, paranitrobenzyl group, paramethoxybenzyl group, andtriphenylmethyl group; ethers such as methoxymethyl group, tertiarybutoxymethyl group, tetrahydropyranyl group, and2,2,2-trichloroethoxymethyl group; (alkyl and/or aralkyl)-substitutedsilyl groups such as trimethylsilyl group, isopropyldimethylsilyl group,tertiary butyldimethylsilyl group, tribenzylsilyl group, and tertiarybutyldiphenylsilyl group.

When more than two of R¹¹, R¹² and R¹³ are protective groups, the actualprotective groups should be chosen is able to be determined according tothe knowledge of the present field of the art to be selectively removedat the synthesis of compound 19 or 20.

The thus produced compound of Example 9 shows strong antibacterialactivity as well as excellent stability and pharmacokinetics as will beevident from the Test Examples as presented below. When this compoundwas evaluated by X ray crystallography, absolute configuration at thepart of the asymmetric carbon at position 7 (the site of the amino groupsubstitution) of 5-azaspiro[2.4]heptane-5-yl group was (7S). Thisconfirmed that the preferable compound is the one in which thespirobicyclic substituent at position 7 has an absolute configuration(S).

Since the compound of the present invention has strong antibacterialactivity, it can be used as a drug for human, animals, and fish, or as apreservative of agricultural chemicals and foods. The typical dose ofthe compound of the present invention when it is used as a human drug is50 mg to 1 g, and more preferably 100 mg to 500 mg per day per adult.When the compound of the invention is administered to an animal, thedose is typically 1 mg to 200 mg, and more preferably 5 mg to 100 mg perday per kg weight of the animal although the dose may vary according tothe size of the animal to be treated, type of the pathogenicmicroorganism, and seriousness of the condition. Such daily dose may beadministered in a single dose or in 2 to 4 divided doses. If necessary,a dose exceeding such daily dose may be administered.

The compound of the present invention has excellent antibacterialactivity for a broad range of microorganisms causing various infections,and therefore, the present compound is capable of treating, preventing,or ameliorating the diseases caused by such pathogenic microorganisms.The compound of the present invention is effective for bacteria and thebacteria-like microorganisms including Staphylococcus, Streptococcuspyogenes, hemolytic streptococcus, enterococcus, pneumococcus,Peptostreptococcus, Neisseria gonorrhoeae, Escherichia coli,Citrobacter, Shigella, Klebsiella pneumoniae, Enterobacter, Serratia,Proteus, Pseudomonas aerugimosa, Haemophilus influenzae, Acinetobacter,Campylobacter, and Chlamydia trachomatis.

The diseases caused by such pathogenic microorganisms includesuperficial secondary infections such as folliculitis, furuncle,carbuncle, erysipelas, cellulitis, lymphangitis, whitlow, subepidermalabscess, hidradenitis, acne conglobata, infectious atheroma, perianalabscess, mastitis, and injury, burn and operative wounds; secondaryinfections of laryngopharyngitis, acute bronchitis, tonsillitis, chromicbronchitis, bronchiectasis, diffuse panbronchiolitis, and chronicrespiratory diseases; pneumonia, pyelonephritis, cystitis, prostatitis,epididymitis, gonorrheal urethritis, nongonococcal urethritis,cholecystitis, cholangitis, shigellosis, enteritis, adnexitis,intrautarine infection, bartholinitis, blepharitis, hordeolum,dacryocystitis, meibomianitis, corneal ulcer, middle otitis, sinusitis,periodontal inflammations, pericoronitis, jaw inflammation, peritonitis,endocarditis, sepsis, meningitis, and skin infections.

The compound of the present invention is also effective for acid fastbacteria such as M. tuberculosis complex (Mycobacterium tuberculosis, M.bovis, and M. africans) and atypical mycobacteria (M. kansasii, M.marianum, M. scrofulaceum, M. avium, M. intracellulare, M. xenopi, M.fortuitum, and M. chelonae). The mycobacterial infections caused by suchpathogenic microorganisms are divided into three categories oftuberculosis, atypical mycobacteriosis, and leprosy. Mycobacterialinfections affect not only the lung but also thoracic cavity, tracheaand bronchus, lymph nodes, by systemic dissemination, joints and bones,meninges and brain, digestive organs (intestine and liver), skin,mammary gland, eyes, auris media and throat, urinary tract, malegenitalia, and female genitalia. The main organ affected by the atypicalmycobacteriosis (non-tuberculous mycobacteriosis) is lung. The atypicalmycobacteriosis, however, also affects by topical lymphadenitis, skinsoft tissues, bones and joints, and by systemic dissemination.

The compound of the present invention is also effective for variousmicroorganisms causing animal infections such as Escherichia,Salmonella, Pasteurella, Haemophilus, Bordetella, Staphylococcus, andmycoplasma. Exemplary diseases include, colibacillosis, pullorumdisease, avian paratyphoid, fowl cholera, infectious diarrhea,staphylococcosis mycoplasma infection, and the like for fowls;colibacillosis, salmonellosis, pasteurellosis, hemophilosis, atrophicrhinitis, exudative epidermitis, mycoplasma infection and the like forpigs; colibacillosis, salmonellosis, hemorrhagic septicemia, mycoplasmainfection, pleuropneumonia, and mastitis for cows; Escherichia colisepsis, salmonella infection, hemorrhagic septicemia, pyometra,cystitis, and the like for dogs; and exudative pleurisy, cystitis,chronic rhinitis, hemophilosis, kitten's diarrhea, mycoplasma infection,and the like for cats.

The antibacterial drug containing the compound of the present inventionmay be prepared by selecting a dosage form appropriate for theadministration rcute, and preparing the drug by a method commonly usedin the art for producing the selected dosage form. Exemplary dosageforms for the antibacterial drug containing the compound of the presentinvention as its main ingredient include tablet, powder, granules,capsule, solution, syrup, elixir, and oil-base and water-basesuspension. In the case of an injection, the preparation may contain astabilizer, an antiseptic, a solubilizer, and the like and thepreparation optionally supplemented with such additives may be filled ina container, and then freeze dried to produce a solid preparation to behydrated immediately before use. The container may be filled either witha single dose or multiple doses. In the case of a solid preparation, thepreparation may contain a pharmaceutically acceptable carrier with thecompound (1), and exemplary carries include fillers, expanders, binders,disintegrants, solubilizers, wetting agents, and lubricants. The liquidpreparation may be a solution, a suspension, an emulsion, or the likewhich may contain a suspending agent or emulsifier as an additive.

In the case of a solid preparation, the preparation may contain apharmaceutically acceptable carrier with the active compound, andexemplary carriers include fillers, binders, disintegrants,solubilizers, wetting agents, and lubricants. The liquid preparation maybe a solution, a suspension, an emulsion, or the like which may containa suspending agent or emulsifier as an additive.

Next, exemplary preparations are described.

Preparation 1 (Capsule) Compound of Example 9 100.0 mg Corn starch 23.0mg Carboxymethlcellulose calcium 22.5 mg Hydroxymethylcellulose 3.0 mgMagnesium stearate 1.5 mg Total 150.0 mg

Preparation 2 (Solution) Compound of Example 9 1 to 10 g Acetic acid orsodium hydroxide 0.5 to 2 g Ethyl paraoxybenzoate 0.1 g Purified water87.9 to 98.4 g Total 100.0 g

Preparation 3 (Powder for animal feed) Compound of Example 9 1 to 10 gCorn starch 89.5 to 98.5 g Light anhydrous silicic acid 0.5 g Total100.0 g

EXAMPLES

Next, the present invention is described in further detail by referringto Reference Examples and Examples which by no means limit the scope ofthe present invention.

Reference Example 1 Ethyl(3R*,4R*)-1-benzyl-3,4-dimethylpyrrolidine-3-carboxylate

To a solution of tiglic acid ethyl ester (6.41 g, 50.0 mmol) andN-benzyl-N-(methoxy-methyl)-N-trimethylsilyl methylamine (15.35 g, 60.0mmol) in dichloromethane (150 mL), a catalytic amount of trifluoroaceticacid was added at room temperature, and the mixture was stirred in anoil bath at 40° C. for 10 hours. The reaction mixture was diluted byadding ethyl acetate (500 mL), and the solution was washed withsaturated aqueous solution of sodium hydrogencarbonate (200 mL) andsaturated aqueous solution of sodium chloride (200 mL), and dried withanhydrous sodium sulfate. After removing the dessicating agent byfiltration, the solvent was removed by distillation under reducedpressure. The resulting residue was purified by silica gel columnchromatography (chloroform : methanol, 49:1→19:1→9:1) to obtain 13.73 gof crude title compound as a pale yellow oil. The crude product was usedin the subsequent reaction with no further purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.96 (3H, d, J=7 .1 Hz), 1.17 (3H, s),1.22 (3H, t, J=7.1 Hz), 2.16 (1H, t, J=8.8 Hz), 2.25 (1H, d, J=9.6 Hz),2.61-2.67 (1H, m), 2.91 (1H, t, J=8.2 Hz), 3.28 (1H, d, J=10.0 Hz), 3.53(1H, d, J=13.5 Hz), 3.64 (1H, d, J=13.2 Hz), 4.11 (2H, q, J=7.1 Hz),7.19-7.38 (5H, m).

MS (ESI) m/z: 262 (M+H)⁺.

Reference Example 2 Ethyl(3R*,4R*)-1-benzyloxycarbonyl-3,4-dimethylpyrrolidine-3-carboxylate

To a solution of ethyl(3R*,4R*)-1-benzyl-3,4-dimethylpyrrolidine-3-carboxylate (2.75 g, 10.0mmol) in dichloromethane (30 mL), benzyl chloroformate (2.14 mL, 15.0mmol) was added at room temperature, and the mixture was stirred at roomtemperature for 6 hours. Benzyl chloroformate (2.14 mL, 15.0 mmol) wasalso added to the mixture, and the mixture was stirred at roomtemperature for another 14 hours. The solvent was removed bydistillation under reduced pressure, and the residue was purified bysilica gel column chromatography (hexane : ethyl acetate, 9:1→4:1→2:1)to obtain 1.64 g of the title compound (5.37 mmol, 2 steps, 54%) as acolorless transparent oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.99 (1.5H, d, J =7.1 Hz), 1.02 (1.5H, d,J=7.1 Hz), 1.18 (3H, s), 1.26 (3H, t, J=7.1 Hz), 2.57-2.66 (1H, m),3.01-3.10 (1H, m), 3.39 (0.5H, d, J=10.7 Hz), 3.45 (0.5H, d, J=11.0 Hz),3.66 (1H, td, J=11.0, 8.0 Hz), 3.77 (1H, dd, J=10.9, 4.8 Hz), 4.11-4.19(2H, m), 5.09-5.17 (2H, m), 7.26-7.38 (5H, m).

MS (ESI) m/z: 306 (M+H)⁺.

Reference Example 3(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine

To a solution of ethyl(3R*,4R*)-1-benzyloxycarbonyl-3,4-dimethylpyrrolidine-3-carboxylate(1.63 g, 5.34 mmol) in ethanol (16 mL), 1N aqueous solution of sodiumhydroxide (16.0 mL, 16.0 mmol) was added at room temperature, and themixture was stirred at room temperature for 3.5 hours. Afterconcentrating the solvent under reduced pressure, 1N hydrochloric acidwas added to the mixture for acidification, and the mixture wasextracted with ethyl acetate (150 mL). The resulting organic layer wasdried with anhydrous sodium sulfate, and after removing the dessicatingagent by filtration, the solvent was removed by distillation underreduced pressure to obtain crude product in the form of carboxylic acid.The crude product in the form of a carboxylic acid was used in thesubsequent reaction with no further purification.

To a solution of the thus obtained crude product in the form of acarboxylic acid and triethylamine (1.488 mL, 10.68 mmol) in toluene (30mL), diphenylphosphoryl azide (1.495 mL, 6.94 mmol) was added in an icebath, and the mixture was stirred at room temperature for 30 minutes,and further stirred in an oil bath at 80° C. for 2 hours. The reactionmixture was diluted by adding ethyl acetate (150 mL), and the solutionwas washed with saturated aqueous solution of sodium hydrogencarbonate(80 mL), water (80 mL), and saturated aqueous solution of sodiumchloride (80 mL) in this order. The resulting organic layer was driedwith anhydrous sodium sulfate, and after removing the dessicating agentby filtration, the solvent was removed by distillation under reducedpressure to obtain crude product in the form of isocyanate. The thusobtained crude product in the form of isocyanate was dissolved in1,4-dioxane (15 mL), and after adding 6N hydrochloric acid (15 mL), themixture was stirred for 1 hour in an oil bath at 50° C. The reactionmixture was concentrated under reduced pressure, and afterazeotropically distilling with ethanol (5 times), the residue wasdissolved in dichloromethane (30 mL), and to this solution at roomtemperature was added triethylamine (3.72 mL, 26.69 mmol), and thendi-tert-butyl dicarbonate (2.33 g, 10.68 mmol). The reaction mixture wasstirred at room temperature for 3 hours, and the solvent was removed bydistillation under reduced pressure. The residue was purified by silicagel column chromatography (hexane : ethyl acetate, 9:1→4:1) to obtain1.10 g (3.16 mmol, 4 steps, 59%) of the title compound as a colorlessgummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.94-0.98 (3H, m), 1.24-1.26 (3H, m)1.42-1.44 (9H, m), 2.44-2.62 (1H, m), 2.99-3.05 (1H, m), 3.63-3.70 (3H,m), 4.54-4.56 (1H, m), 5.08-5.17 (2H, m), 7.28-7.37 (5H, m)

MS (ESI) m/z: 371 (M+Na)⁺.

Reference Example 4(+)-(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidineand(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine

The racemic compound of(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(1.10 g, 3.16 mmol) produced in Reference Example 3 was opticallyresolved by using an optically active column (CHIRALPAK AD, 20 mmdiam.×250 mm; hexane : isopropyl alcohol, 95:5; flow rate, 25 mL/minute;resolution, 30 mg per run) to produce(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(528 mg; 1.52 mmol; retention time=12.8 minutes, [α]D25.1=+8.1°(c=0.161, chloroform)) and(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(532 mg; 1.53 mmol; retention time=15.8 minutes; [α]D25.1=−6.3°(c=0.175, chloroform)).

Example 17-[(3R*,4S*)-3-Amino-3,4-dimethylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

To a solution of(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(490 mg, 1.406 mmol) in methanol (20 mL) was added 10% palladium-carboncatalyst (M; water content, about 50%; 147 mg), and the suspension wasstirred in hydrogen atmosphere at room temperature for 2 hours. Afterremoving the catalyst by filtration, the solvent was removed bydistillation under reduced pressure to obtain crude product (314 mg,quantitative) of(3R*,4S*)-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine as acolorless gummy solid.

The thus obtained(3R*,4S*)-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine crudeproduct (314 mg),6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (461 mg, 1.277 mmol), and triethylamine(0.534 mL, 3.83 mmol) were dissolved in dimethyl sulfoxide (4 mL), andthe mixture was stirred in an oil bath at 35° C. for 18 hours. To thisreaction mixture were added a mixed solution of ethanol and water(ethanol : water=4:1) (20 mL) and triethylamine (2 mL), and the mixturewas heated under reflux in an oil bath at 100° C. for 2 hours. Afterconcentrating the reaction mixture under reduced pressure, the residuewas dissolved in ethyl acetate (150 mL), and washed with 10% aqueoussolution of citric acid (80 mL), water (80 mL×2), and saturated aqueoussolution of sodium chloride (80 mL). The organic layer was dried withanhydrous sodium sulfate, and the solvent was removed by distillationunder reduced pressure. The residue was dissolved in concentratedhydrochloric acid (20 mL) in an ice bath, and solution was stirred atroom temperature for 10 minutes. The reaction mixture was washed withchloroform (30 mL×3). To the aqueous layer was added 10 mol/l aqueoussolution of sodium hydroxide in an ice bath to adjust the pH to 12.0,and the solution was further adjusted to pH 7.4 with hydrochloric acid.The solution was then extracted with a mixed solution of chloroform andmethanol (chloroform : methanol=9:1) (150 mL×2). The organic layer wasdried with anhydrous sodium sulfate, and the solvent was removed bydistillation under reduced pressure. The residue was purified byrecrystallization from ethanol, and the crystals were dried underreduced pressure to obtain the title compound 328 mg (0.805 mmol, 63%)as a pale yellow powder.

mp: 200-203° C.

[α]_(D) ^(25.1)=+213.7° (c=0.204, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.00 (3H, d, J=6.6 Hz), 1.12 (3H, s),1.55-1.70 (2H, m), 2.07 (1H, m), 3.39 (1H, d, J=10.0 Hz), 3.48-3.69 (6H,m), 4.04 (1H, m), 4.93 (1H, dd, J=39.1, 1.5 Hz), 7.64 (1H, d, J=14.6Hz), 8.47 (1H, s).

Elementary analysis for C₂₀H₂₃F₂N₃O₄.1.5H₂O:

Calculated: C, 55.29; H, 6.03; F, 8.75; N, 9.67.

Found: C, 55.55; H, 6.03; F, 8.45; N, 9.56.

MS (FAB) m/z: 408 (M+H)⁺.

IR (ATR): 2974, 2935, 2879, 1722, 1614, 1572, 1537, 1502, 1456, 1390,1356, 1323, 1271, 1207 cm⁻¹.

Example 27-[(3R*,4S*)-3-Amino-3,4-dimethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(480 mg, 1.378 mmol) was converted to(3R*,4S*)-3-(tert-butoxycarbonylamino-3,4-dimethylpyrrolidine crudeproduct (311 mg, quantitative), and the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (452 mg, 1.252 mmol) to obtain 348 mg (0.854mmol, 68%) of the title compound as a pale yellow powder.

mp: 195-196° C.

[α]_(D) ^(25.1)=−118.3° (c=0.224, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.00 (3H, d, J=6.6 Hz), 1.14 (3H, s),1.31-1.44 (1H, m), 1.49-1.59 (1H, m), 2.09 (1H, m), 3.39-3.57 (6H, m),3.71 (1H, m), 3.97-4.02 (1H, m), 5.00 (1H, dm, J=63.7 Hz), 7.63 (1H, d,J=14.6 Hz), 8.39 (1H, d, J=2.4 Hz).

Elementary analysis for C₂₀H₂₃F₂N₃O₄.0.75H₂O:

Calculated: C, 57.07; H, 5.87; F, 9.03; N, 9.98.

Found: C, 57.30; H, 5.90; F, 9.13; N, 9.92.

MS (FAB) m/z: 408 (M+H)⁺.

IR (ATR): 2962, 2873, 1724, 1616, 1513, 1435, 1362, 1321, 1271 cm⁻¹.

Reference Example 5 Methyl(3R*,4S*)-1-benzyl-3,4-dimethylpyrrolidine-3-carboxylate

The procedure of Reference Example 1 was repeated by using methylangelate (12.01 mL, 100.0 mmol) and N-benzyl-N-(n-butoxymethyl)-N-trimethylsilyl methylamine (36.0 g, 128.9 mmol) to obtain12.28 g of the crude title compound as a yellow oil. The thus obtainedcrude product was used in the subsequent reaction with no furtherpurification.

MS (ESI) m/z: 248 (M+H)⁺.

Reference Example 6 Methyl(3R*,4S*)-1-benzyloxycarbonyl-3,4-dimethylpyrrolidine-3-carboxylate

The procedure of Reference Example 2 was repeated by using the thussynthesized methyl(3R*,4S*)-1-benzyl-3,4-dimethylpyrrolidine-3-carboxylate crude product(12.28 g) and benzyl chloroformate (21.3 mL, 149.3 mmol) to obtain 4.23g (14.52 mmol, 2 steps, 15%) of the title compound as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.94 (1.5H, d, J=6.8 Hz), 0.96 (1.5H, d,J=6.7 Hz), 1.30 (1.5H, s), 1.31 (1.5H, s), 2.14 (1H, m), 3.16-3.28 (2H,m), 3.64-3.71 (4H, m), 3.92 (1H, dd, J=14.6, 11.5 Hz), 5.16 (2H, m),7.26-7.37 (5H, m).

MS (ESI) m/z: 292 (M+H)⁺.

Reference Example 7(3R*,4R*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine

To a solution of methyl(3R*,4S*)-1-benzyloxycarbonyl-3,4-dimethylpyrrolidine-3-carboxylate(4.23 g, 14.52 mmol) in methanol (88 mL), 1N aqueous solution of sodiumhydroxide (44.0 mL, 44.0 mmol) was added at room temperature, and themixture was stirred at room temperature for 5 hours, and in an oil bathat 50° C. for another 19 hours. To the mixture was added sodiumhydroxide (1.742 g, 43.6 mmol), and this mixture was also stirred in anoil bath at 50° C. for 8 hours. After concentrating the solvent underreduced pressure, concentrated hydrochloric acid was added to theconcentrate in an ice bath for acidification, and the solution wasextracted with ethyl acetate (300 mL). The resulting organic layer wasdried with anhydrous sodium sulfate, and the dessicating agent wasremoved by filtration. The solvent was removed by distillation underreduced pressure to obtain the crude product in the form of a carboxylicacid. The crude product in the form of a carboxylic acid was used in thesubsequent reaction with no further purification.

To a solution of the thus obtained crude product in the form of acarboxylic acid and triethylamine (6.06 mL, 43.5 mmol) in toluene (70mL), diphenylphosphoryl azide (4.06 mL, 18.84 mmol) was added in an icebath, and the mixture was stirred at room temperature for 2 hours, andin an oil bath at 90° C. for another 1 hour. To the heated and stirredreaction mixture was added tert-butyl alcohol (70 mL), and the mixturewas heated under reflux in an oil bath at 120° C. for 93 hours, and thereaction mixture was then cooled to room temperature. After removing thesolvent by distillation under reduced pressure, the residue was purifiedby silica gel column chromatography (hexane-ethyl acetate90:10→85:15→80:20→75:25) to obtain 1.205 g (3.46 mmol, 2 steps, 24%) ofthe title compound as a colorless gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.99 (1.5H, d, J=7.3 Hz), 1.01 (1.5H, d,J=7.1 Hz), 1.43-1.46 (12H, m), 2.06-2.22 (1H, m), 3.11-3.17 (1H, m),3.24 (0.5H, d, J=11.5 Hz), 3.30 (0.5H, d, J=11.2 Hz), 3.60-3.67 (1H, m),3.88 (0.3H, d, J=11.2 Hz), 4.02 (0.5H, d, J=11.0 Hz), 4.43 (1H, brs),5.09-5.17 (2H, m), 7.26-7.37 (5H, m).

MS (ESI) m/z: 293 (M-tBu)⁺.

Reference Example 8(+)-(3R*,4R*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidineand(−)-(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine

The racemic compound of(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(1.205 g, 3.46 mmol) produced in Reference Example 7 was opticallyresolved in an optically active column (CHIRALPAK AS, 20 mm diam.×250mm; hexane : isopropyl alcohol, 95:5; flow rate, 20 mL/minute;resolution, 40 mg per run) to obtain(+)-(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(468 mg, 1.34 mmol, retention time=9.0 minutes, [α]D25.1=+10.3°(c=0.165, chloroform)) and(−)-(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(591 mg, 1.70 mmol, retention time=11.4 minutes, [α]D25.1=−12.0°(c=0.150, chloroform)).

Example 37-[(3R*,4R*)-3-Amino-3,4-dimethylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(+)-(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(468 mg, 1.343 mmol) was converted to(3R*,4R*)-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine crudeproduct (280 mg, 1.307 mmol, 97%), and the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid -difluoroboron complex (429 mg, 1.188 mmol) to obtain 370 mg (0.834mmol, 68%) of the title compound as a white powder.

mp: 175-179° C.

[α]_(D) ^(23.8)=−107.1° (c=0.240, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.00 (3H, d, J=6.8 Hz), 1.25 (3H, s),1.31-1.43 (1H, m), 1.48-1.59 (1H, m), 2.03-2.13 (1H, m), 3.41 (1H, dd,J=10.5, 1.7 Hz), 3.46-3.52 (4H, m), 3.62-3.70 (2H, m), 3.99 (1H, dt,J=10.2, 4.5 Hz), 5.01 (1H, ddd, J=63.8, 8.7, 5.5 Hz), 7.63 (1H, d,J=14.6 Hz), 8.39 (1H, d, J=2.9 Hz).

Elementary analysis for C₂₀H₂₃F₂N₃O₄.2H₂O:

Calculated: C, 54.17; H, 6.14; F, 8.57; N, 9.48.

Found: C, 54.41; H, 5.81; F, 8.63; N, 9.37.

MS (EI) m/z: 407 (M⁺).

IR (ATR): 2962, 2881, 2833, 1726, 1614, 1577, 1510, 1435, 1387, 1354,1306, 1267 cm⁻¹.

Example 47-[(3R*,4R*)-3-Amino-3,4-dimethylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(−)-(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine(169 mg, 0.485 mmol) was converted to(3R*,4R*)-3-(tert-butoxycarbonylamino)-3,4-dimethylpyrrolidine crudeproduct (95 mg, 0.443 mmol, 91%), and the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid -difluoroboron complex (145 mg, 0.402 mmol) to obtain 65 mg (0.146mmol, 36%) of the title compound as a white powder.

mp: 209-211° C.

[α]_(D) ^(23.7)=+186.0° (c=0.162, 0.1N NaCH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.01 (3H, d, J=6.8 Hz), 1.26 (3H, s),1.53-1.71 (2H, m), 2.10 (1H, m), 3.34 (1H, d, J=7.5 Hz), 3.54-3.62 (5H,m), 3.76 (1H, dd, J=10.5, 2.9 Hz), 4.05 (1H, m), 4.80-5.02 (1H, m), 7.64(1H, d, J=14.6 Hz), 8.48 (1H, s).

Elementary analysis for C₂₀H₂₃F₂N₃O₄.0.5EtOH.0.75H₂O:

Calculated: C, 56.81; H, 6.24; F, 8.56; N, 9.46.

Found: C, 56.72; H, 6.26; F, 8.44; N, 9.30.

MS (EI) m/z: 407 (M⁺).

IR (ATR): 2964, 2870, 2833, 1726, 1616, 1577, 1537, 1495, 1456, 1392,1358, 1298, 1265, 1203 cm⁻¹.

Reference Example 9 Methyl(3R*,4R*)-1-benzyl-4-ethyl-3-methylpyrrolidine-3-carboxylate

The procedure of Reference Example 1 was repeated by using methyltrans-2-methyl-2-pentenate (2.70 g, 21.1 mmol) and N-benzyl-N-(methoxymethyl)-N-trimethylsilyl methylamine (5.00 g, 21.1 mmol) to obtain 3.70g (14.06 mmol, 67%) of the title compound as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.81 (3H, t, J=7.4 Hz), 1.14 (3H, s),1.15-1.24 (1H, m), 1.44-1.54 (1H, m), 2.16 (1H, t, J=9.1 Hz), 2.22 (1H,d, J=9.6 Hz), 2.38-2.46 (1H, m), 2.92 (1H, dd, J=8.8, 7.8 Hz), 3.21 (1H,d, J=9.3 Hz), 3.51 (1H, d, J=13.2 Hz), 3.62 (3H, s), 3.62 (1H, d, J=13.2Hz), 7.15-7.27 (5H, m).

MS (ESI) m/z: 262 (M+H)⁺.

Reference Example 10 Methyl(3R*,4R*)-1-benzyloxycarbonyl-4-ethyl-3-methylpyrrolidine-3-carboxylate

The procedure of Reference Example 2 was repeated by using methyl(3R*,4R*)-1-benzyl-4-ethyl-3-methylpyrrolidine-3-carboxylate (3.68 g,14.08 mmol) to obtain 3.68 g (12.05 mmol, 86%) of the title compound asa colorless transparent oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.86-0.92 (3H, m), 1.18 (3H, s),1.23-1.34 (1H, m), 1.51-1.59 (1H, m), 2.42-2.54 (1H, m), 3.06 (1H, m),3.40 (0.5H, d, J=10.7 Hz), 3.47 (0.5H, d, J=11.0 Hz), 3.68-3.79 (5H, m),5.09-5.19 (2H, m), 7.28-7.38 (5H, m).

MS (ESI) m/z: 306 (M+H)⁺.

Reference Example 11(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine

The procedure of Reference Example 3 was repeated by using methyl(3R*,4R*)-1-benzyloxycarbonyl-4-ethyl-3-methylpyrrolidine-3-carboxylate(3.68 g, 12.05 mmol) to obtain 3.25 g (8.97 mmol, 4 steps, 74%) of thetitle compound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.91-0.98 (3H, m), 1.23-1.31 (4H, m)1.41-1.54 (10H, m), 2.25-2.42 (1H, m), 2.99-3.06 (1H, m), 3.57-3.75 (3H,m), 4.55-4.59 (1H, m), 5.08-5.17 (2H, m), 7.27-7.38 (5H, m).

MS (ESI) m/z: 307 (M-tBu)⁺.

Reference Example 12(+)-(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidineand(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine

The racemic body of(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine(800 mg, 2.21 mmol) produced in Reference Example 11 was opticallyresolved in an optically active column (CHIRALPAK IA, 20 mm diam.×250mm; hexane : dichloromethane, 75:25; flow rate, 20 mL/minute;resolution, 10 mg per run) to obtain(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarborylamino)-4-ethyl-3-methylpyrrolidine(393 mg, 1.084 mmol, retention time=11.3 minutes, [α]D25.1=+15.2°(c=0.230, chloroform)) and(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine(396 mg, 1.093 mmol, retention time=13.1 minutes, [α]D25.1=−10.4(c=0.125, chloroform)).

Example 57-[(3R*,4S*)-3-Amino-4-ethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine(383 mg, 1.057 mmol) was converted to crude(3R*,4S*)-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine, andthe product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid-difluoroboron complex (361 mg, 1.000 mmol) to obtain 260 mg (0.618mmol, 62%) of the title compound as a white powder.

mp: 209-211° C.

[α]_(D) ^(25.1)=−154.2° (c=0.144, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.94 (3H, t, J=7.3 Hz), 1.14 (3H, s),1.19-1.41 (2H, m), 1.53 (1H, m), 1.61-1.67 (1H, m), 1.88-1.95 (1H, m),3.40-3.42 (1H, m), 3.47-3.53 (1H, m), 3.54 (3H, s), 3.60 (1H, dd,J=10.0, 3.4 Hz), 3.73 (1H, t, J=9.2 Hz), 3.96-4.01 (1H, m), 5.02 (1H,ddd, J=63.8, 8.5, 5.4 Hz), 7.64 (1H, d, J=14.4 Hz), 8.38 (1H, d, J=3.2Hz).

Elementary analysis for C₂₁H₂₅F₂N₃O₄.0.5H₂O:

Calculated: C, 58.60; H, 6.09; F, 8.83; N, 9.76.

Found: C, 58.68; H, 5.94; F, 9.03; N, 9.69.

MS (FAB) m/z: 422 (M+H)⁺.

IR (ATR): 2964, 2931, 2875, 1716, 1618, 1514, 1448, 1439, 1371, 1325,1279, 1234 cm⁻¹.

Example 67-[(3R*,4S*)-3-Amino-4-ethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine(386 mg, 1.065 mmol) was converted to crude(3R*,4S*)-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine, andthe product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (361 mg, 1.000 mmol) to obtain 263 mg (0.625mmol, 63%) of the title compound as a white powder.

mp: 113-115° C.

[α]_(D) ^(25.1)=+234.5° (c=0.310, 0.1N NaOE)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.91 (3H, t, J=7.3 Hz), 1.09 (3H, s),1.17-1.28 (1H, m), 1.52-1.70 (3H, m), 1.80-1.88 (1H, m), 3.31 (1H, d,J=9.8 Hz), 3.49-3.53 (5H, m), 3.63-3.69 (1H, m), 4.02 (1H, m), 4.80-4.98(1H, m), 7.64 (1H, d, J=14.6 Hz), 8.48 (1H, s).

Elementary analysis for C₂₁H₂₅F₂N₃O₄.0.25EtOH.0.5H₂O:

Calculated: C, 58.43; H, 6.27; F, 8.60; N, 9.51.

Found: C, 58.36; H, 6.26; F, 8.68; N, 9.49.

MS (FAB) m/z: 422 (M+H)⁺.

IR (ATR): 2960, 2929, 2873, 1728, 1614, 1579, 1541, 1510, 1433, 1392,1352, 1296, 1275 cm⁻¹.

Reference Example 13 Ethyl Trans-4-fluoro-2-methyl-2-butenate

To a solution of ethyl trans-4-hydroxy-2-methyl-2-butenate synthesizedby the method of Wolff, M. (Tetrahedron Letters, vol. 43, pages2555-2559, 2002) (2.73 g, 18.94 mmol) in dichloromethane (100 mL),diethylaminosulfur trifluoride (7.45 mL, 56.9 mmol) was added dropwisein an ice bath in 10 minutes, and the mixture was stirred at the sametemperature for 2 hours. To the reaction mixture was added saturatedaqueous solution of sodium hydrogencarbonate (80 mL), and the solutionwas extracted with dichloromethane (200 mL+2×100 mL). The organic layerwas dried with anhydrous sodium sulfate, and the dessicating agent wasremoved by using a short silica gel column. The solvent was removed bydistillation under reduced pressure to obtain 2.43 g (16.63 mmol, 88%)of the target compound as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.31 (3H, t, J=7.2 Hz), 1.86-1.87 (3H,m), 4.22 (2H, q, J=7.2 Hz), 5.09 (2H, ddd, J=46.5, 5.9, 1.1 Hz),6.82-6.89 (1H, m).

Reference Example 14 Ethyl(3R*,4R*)-1-benzyl-4-fluoromethyl-3-methylpyrrolidine-3-carboxylate

The procedure of Reference Example 1 was repeated by using ethyltrans-4-fluoro-2-methyl-2-butenate (2.43 g, 16.63 mmol) andN-benzyl-N-(methoxy methyl)-N-trimethylsilyl methylamine (5.11 mL, 19.97mmol) to obtain 2.57 g (9.20 mmol, 55%) of the title compound as a paleyellow.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.25 (3H, t, J=7.2 Hz), 1.30 (3H, s),2.33 (1H, d, J=9.3 Hz), 2.46 (1H, dd, J=9.0, 7.1 Hz), 2.89-3.06 (2H, m),3.16 (1H, d, J=9.3 Hz), 3.57 (1H, d, J=13.2 Hz), 3.64 (1H, d, J=13.2Hz), 4.16 (2H, ddd, J=14.3, 7.1, 2.6 Hz), 4.44 (1H, ddd, J=34.9, 9.3,6.0 Hz), 4.56 (1H, ddd, J=34.7, 9.3, 6.1 Hz), 7.21-7.35 (5H, m).

MS (ESI) m/z: 280 (M+H)⁺.

Reference Example 15 Ethyl(3R*,4R*)-1-benzyloxycarbonyl-4-fluoromethyl-3-methylpyrrolidine-3-carboxylate

The procedure of Reference Example 2 was repeated by using ethyl(3R*,4R*)-1-benzyl-4-fluoromethyl-3-methylpyrrolidine-3-carboxylate(2.56 g, 9.16 mmol) to obtain 2.56 g (7.92 mmol, 86%) of the titlecompound as a colorless transparent oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.26 (3H, t, J=7.2 Hz), 1.29 (3H, s),2.91-3.00 (1H, m), 3.35-3.48 (2H, m), 3.71-3.77 (1H, m), 3.81 (1H, d,J=11.0 Hz), 4.11-4.21 (2H, m), 4.41-4.68 (2H, m), 5.10-5.18 (2H, m),7.29-7.37 (5H, m).

MS (ESI) m/z: 324 (M+H)⁺.

Reference Example 16(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine

The procedure of Reference Example 3 was repeated by using ethyl(3R*,4R*)-1-benzyloxycarbonyl-4-fluoromethyl-3-methylpyrrolidine-3-carboxylate(2.55 g, 7.89 mmol) to obtain 2.14 g (5.84 mmol, 4 steps, 74%) of thetitle compound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.37 (1.8H, s), 1.39 (1.2H, s), 1.42 (9H,m), 2.79-3.13 (1H, m), 3.19-3.31 (1H, m), 3.60-3.72 (3H, m), 4.41-4.62(2H, m), 4.77 (0.4H, brs), 4.85 (0.6H, brs), 5.09-5.17 (2H, m),7.28-7.37 (5H, m).

MS (ESI) m/z: 311 (M-tBu)⁺.

Reference Example 17(−)-(3R*,4S*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidineand(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine

The racemic body of(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine (1.454 g, 3.97 mmol) produced inReference Example 16 was optically resolved in an active column(CHIRALPAK AS, 20 mm diam.×250 mm; hexane : isopropyl alcohol, 93:7;flow rate, 215 mL/minute; resolution, 60 mg per run) to obtain(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine (624 mg, 1.703 mmol, retentiontime=11.8 minutes, [α]D25.1=−15.0° (c=0.645, chloroform)) and(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine (623 mg, 1.700 mmol, retentiontime=15.5 minutes, [α]D25.1=+13.8° (c=1.230, chloroform)).

Example 77-[(3R*,4S*)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(−)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(303 mg, 0.827 mmol) was converted to crude(3R*,4S*)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine,and the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (299 mg, 0.827 mmol) to obtain 231 mg (0.521mmol, 63%) of the title compound as a white powder.

mp: 195-198° C.

[α]_(D) ^(25.1)=−36.8° (c=0.125, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.29 (3H, s), 1.45 (1H, d, J=27.2Hz), 1.52-1.64 (1H, m), 2.50 (1H, td, J=13.5, 6.9 Hz), 3.54-3.57 (2H,m), 3.58 (3H, s), 3.63 (1H, t, J=9.2 Hz), 3.94 (1H, t, J=9.2 Hz),3.99-4.05 (1H, m), 4.63 (1H, ddd, J=37.6, 9.2, 6.5 Hz), 4.98 (2H, d,J=64.2 Hz), 7.66 (1H, d, J=14.5 Hz), 8.42 (1H, d, J=2.0 Hz).

Elementary analysis for C₂₀H₂₂F₃N₃O₄.1H₂O):

Calculated: C, 54.17; H, 5.46; F, 12.85; N, 9.48.

Found: C, 54.34; H, 5.41; F, 13.13; N, 9.21.

MS (EI) m/z: 426 (M+H)⁺.

IR (ATR): 3541, 3089, 2972, 2881, 1716, 1622, 1514, 1456, 1365, 1327,1279, 1238 cm⁻¹.

Example 87-[(3R*,4S*)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1,(+)-(3R*,4S*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(310 mg, 0.846 mmol) was converted to crude(3R*,4S*)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine,and the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (305 mg, 0.846 mmol) to obtain 257 mg (0.516mmol, 61%) of the title compound as a white powder.

mp: 186-189° C.

[α]_(D) ^(25.1)=+132.0° (c=0.103, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.28 (3H, s), 1.51-1.70 (2H, m),2.45-2.59 (1H, m), 3.46 (1H, d, J=9.8 Hz), 3.60 (3H, s), 3.66 (2H, q,J=7.1 Hz), 3.69 (1H, dd, J=10.0, 3.2 Hz), 3.74-3.79 (2H, m), 4.03-4.09(1H, m), 4.66 (1H, ddd, J=37.3, 9.6, 6.4 Hz), 4.93 (2H, d, J=71.6 Hz),7.67 (1H, d, J=14.5 Hz), 8.47 (1H, s).

Elementary analysis for C₂₀H₂₂F₃N₃O₄. 1EtOH.1.5H₂O:

Calculated: C, 53.01; H, 6.27; F, 11.43; N, 8.43.

Found: C, 53.03; H, 6.02; F, 11.86; N, 8.08.

MS (EI) m/z: 426 (M+H)⁺.

IR (ATR): 2970, 2883, 1728, 1616, 1560, 1456, 1390, 1350, 1336, 1315,1298, 1267, 1203 cm⁻¹.

Reference Example 18 tert-Butyl 1-acetyl-1-cyclopropanecarboxylate

A mixture of tert-butyl acetoacetate (497 mL, 3.00 mol),1,2-dibromoethane (310 mL, 3.60 mmol), potassium carbonate (1.106 k g,8.00 mmol), and dimethylformamide (2.0 L) was stirred in a water bath at30° C. for 1.5 hours, in a water bath at 60° C. for 3.5 hours, and in awater bath at 30° C. for 4 days. The reaction mixture was separated byfiltration through celite, and the residue on the celite was washed withdiethylether (3.5 L). The filtrate and the diethylether used for thewashing were combined and added to water (2 L), and the organic layerwas separated. The aqueous layer was extracted with diethylether (2 L),and water (1 L) was added to the resulting aqueous layer, and furtherextraction was conducted by adding diethylether (2 L). All organiclayers were combined, and washed with 10% aqueous solution of citricacid (2 L), water (2 L×3), and saturated aqueous solution of sodiumchloride (2 L×3), and dried by adding anhydrous sodium sulfate. Afterremoving the dessicating agent by filtration, the solvent was removed bydistillation under reduced pressure, and the residue was distilled underreduced pressure to obtain 371.8 g of the target compound (10 mmHg,distillation fraction of 72 to 78° C., 2.02 mol, 67%) as a colorlesstransparent oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.37-1.40 (4H, m), 1.49 (9H, s), 2.44(3H, s).

Reference Example 19 tert-Butyl1-(1-amino-1-cyanoethyl)-1-cyclopropanecarboxylate

tert-Butyl 1-acetyl-1-cyclopropanecarboxylate (9.21 g, 50.0 mmol) wasdissolved in a 7N solution of ammonia in methanol (300 mL), and to thissolution in an ice bath were added concentrated ammonia solution (90mL), ammonium chloride (53.5 g, 1.00 mol), and sodium cyanide (4.90 g,100.0 mmol). The mixture was stirred at room temperature for 18 hours,and the solvent was concentrated under reduced pressure. To theconcentrate was added water (100 mL), and the solution was extractedwith dichloromethane (300 mL+2×100 mL). The organic layers werecombined, and dried by adding anhydrous sodium sulfate. After removingthe dessicating agent by filtration, the solvent was removed bydistillation under reduced pressure to obtain 10.15 g (48.3 mmol, 97%)of the crude target compound as a pale brown oil. The thus obtainedcrude product was used in the subsequent reaction with no furtherpurification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.02-1.12 (2H, m), 1.19-1.17 (2H, m),1.48 (9H, s), 1.50 (3H, s), 2.13 (2H, brs).

MS (ESI) m/z: 155 (M-tBu)⁺.

Reference Example 20 tert-Butyl1-(1,2-diamino-1-methylethyl)-1-cyclopropane carboxylate

To a solution (50 mL) of tert-butyl1-(1-amino-1-cyanoethyl)-1-cyclopropanecarboxylate (1.12 g, 5.30 mmol)in ethanol was added a suspension (30 mL) of Raney nickel catalyst(R-100 manufactured by Nikko Rica Corporation, 10 mL) in ethanol, andthe suspension was vigorously stirred at room temperature for 6 hours inhydrogen gas atmosphere. The catalyst was removed by filtration throughcelite, and the solvent was removed by distillation under reducedpressure to obtain 0.84 g (3.92 mmol, 74%) of the crude target compoundas a colorless transparent oil. The thus obtained crude product was usedin the subsequent reaction with no further purification.

MS (ESI) m/z: 215 (M+H)⁺.

Reference Example 211-(1,2-Diamino-1-methylethyl)-1-cyclopropanecarboxylic aciddihydrochloride

0.82 g (3.83 mmol) of the crude tert-butyl 1-(1,2-diamino-1-methylethyl)-1-cyclopropanecarboxylate was dissolved in concentratedhydrochloric acid (5 mL) at room temperature, and the solution wasstirred at the same temperature for 30 minutes. After adding water tothe reaction solution, the solvent was removed by distillation underreduced pressure. The residue was azeotropically distilled with ethanol(twice) to obtain 0.82 g (3.55 mmol, 93%) of the crude target compoundas a pale yellow foam solid. The thus obtained crude product was used inthe subsequent reaction with no further purification.

¹H-NMR (400 MHz, CD₃OD) δ ppm: 1.20-1.26 (1H, m), 1.28 (3H, s),1.32-1.43 (2H, m), 1.58-1.62 (1H, m), 3.46 (1H, d, J=13.4 Hz), 3.80 (1H,d, J=13.4 Hz).

MS (ESI) m/z: 159 (M+H)⁺.

Reference Example 227-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one

To a solution (70 mL) of the crude 1-(1,2-diamino-1-methylethyl)-1-cyclopropanecarboxylic acid dihydrochloride (800 mg, 3.46 mmol)in acetonitrile was added 1,1,1,3,3,3-hexamethyldisilazane (7.38 mL,34.6 mmol), and the mixture was heated under reflux in an oil bath at100° C. for 4 hours under nitrogen atmosphere. The mixture was cooled toroom temperature, and after adding methanol (70 mL), the solvent wasremoved by distillation under reduced pressure to obtain crude7-amino-7-methyl-5-azaspiro[2.4]heptan-4-one as a pale brown gummysolid.

MS (ESI) m/z: 141 (M+H)⁺.

To the thus obtained crude 7-amino-7-methyl-5-azaspiro[2.4]heptan-4-onewere added 1,4-dioxane (20 mL) and di-tert-butyl dicarbonate (1.528 g,7.00 mmol) at room temperature, and the mixture was stirred at thetemperature for 5 hours. Water (50 mL) was added to this reactionmixture, and the mixture was extracted with chloroform (100 mL+50 mL).The organic layers were combined and dried with anhydrous sodiumsulfate. The dessicating agent was removed by filtration through a shortsilica gel column, and the solvent was removed by distillation underreduced pressure. Diethylether was added to the residue, and theresulting suspension was filtered to obtain 502 mg (2.09 mmol, 2 steps,60%) of the target compound as a white powder.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.77-0.82 (1H, m), 0.94-1.04 (2H, m)1.16-1.23 (1H, m), 1.28 (3H, s), 1.43 (9H, s), 3.29 (1H, d, J=10.3 Hz),4.12 (1H, m), 4.60 (1H, brs), 5.82 (1H, brs).

MS (ESI) m/z: 185 (M-tBu)⁺.

Reference Example 235-Benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one

To a solution (65 mL) of7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one (3.12g, 12.97 mmol) in dimethylformamide in an ice bath was gradually addedsodium hydride (55%, dispersion in a mineral oil, 538 mg, 12.33 mmol) in5 minutes, and the mixture was stirred at the same temperature for 40minutes. Benzyl bromide (1.851 mL, 15.56 mmol) was added, and themixture was stirred at room temperature for 1.5 hours. The reactionmixture was diluted by adding ethyl acetate (300 mL), and the solutionwas washed with water (100 mL×2) and saturated aqueous solution ofsodium chloride (100 mL). After drying the solution with anhydroussodium sulfate and removing the dessicating agent by filtration, thesolvent was removed by distillation under reduced pressure. The residuewas purified by silica gel column chromatography (hexane : ethylacetate, 9:1→4:1→2:1) to obtain 4.20 g (12.71 mmol, 98%) of the targetcompound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.76-0.81 (1H, m), 0.93-1.06 (2H, m),1.21-1.29 (4H, m), 1.37 (9H, m), 3.14 (1H, d, J=10.3 Hz), 3.92-3.98 (1H,m), 4.44 (1H, d, J=15.1 Hz), 4.56 (1H, d, J=14.6 Hz), 4.56 (1H, brs),7.22-7.33 (5H, m).

MS (ESI) m/z: 331 (M+H)⁺.

Reference Example 24(−)-5-Benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-oneand(+)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one

The racemic body of5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-oneproduced in Reference Example 23 (2.254 g, 6.82 mmol) was opticallyresolved in an optically active column (CHIRALPAK AD, 20 mm diam.×250mm; hexane: isopropyl alcohol, 90:10; flow rate, 20 mL/minute;resolution, 50 mg per run) to obtain(−)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one(997 mg, 3.02 mmol, retention time=7.0 minutes, [α]_(D)^(25.1)=−113.9°(c=0.180, chloroform)) and(+)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one(957 mg, 2.90 mmol, retention time=11.3 minutes, [α]_(D)=+108.8°(c=0.249, chloroform)).

Reference Example 25(−)-5-Benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane

To a solution (15 mL) of(−)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one(950 mg, 2.88 mmol) in dichloromethane at room temperature was addedtrifluoroacetic acid (7.5 mL), and the mixture was stirred at the sametemperature for 40 minutes. The solvent was removed by distillationunder reduced pressure. After azeotropically distilling the solutionwith toluene (twice), saturated aqueous solution of sodiumhydrogencarbonate (30 mL) was added, and the solution was extracted withchloroform (100 mL+2×50 mL). The organic layers were combined, and driedwith anhydrous sodium sulfate. After removing the dessicating agent byfiltration, the solvent was removed by distillation under reducedpressure. The residue was dissolved in tetrahydrofuran (30 mL), andwhile stirring the solution in an ice bath. Lithium aluminum hydride(218 mg, 5.74 mmol) was added to the solution, and the solution wasstirred at the same temperature for 1 hour. After adding another portionof lithium aluminum hydride (109 mg, 2.87 mmol) and stirring thesolution at room temperature for 2.5 hours, the solution was ice cooled,and water (0.31 mL), 15% aqueous solution of sodium hydroxide (0.31 mL),and water (0.93 mL) were carefully added in this order. The resultingmixture was stirred overnight at room temperature, dried with magnesiumsulfate, and subjected to filtration through celite. The filtrate wasconcentrated under reduced pressure to obtain the crude7-amino-5-benzyl-7-methyl-5-azaspiro[2.4]heptane as a colorlesstransparent oil. The thus obtained crude product was used in thesubsequent reaction with no further purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.37-0.45 (2H, m), 0.56-0.66 (2H, m),0.96 (3H, s), 2.48 (1H, d, J=9.0 Hz), 2.55 (1H, d, J=8.8 Hz), 2.74 (2H,d, J=9.0 Hz), 3.59 (2H, s), 7.21-7.37 (5H, m).

MS (ESI) m/z: 217 (M+H)⁺.

The crude 7-amino-5-benzyl-7-methyl-5-azaspiro[2.4]heptane was dissolvedin dichloromethane (15 mL), and after adding di-tert-butyl dicarbonate(1.255 g, 5.75 mmol), the mixture was stirred at room temperature for 22hours. The solvent was removed by distillation under reduced pressure,and the residue was purified by silica gel column chromatography(chloroform : methanol : triethylamine, 98:2:1→95:5:1) to obtain 586 mg(1.852 mmol, 3 steps, 64%) of the target compound as a colorlesstransparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.40-0.45 (1H, m), 0.50-0.55 (1H, m),0.63-0.69 (1H, m), 0.80-0.85 (1H, m), 1.20 (3H, s), 1.43 (9H, s), 2.44(1H, d, J=8.8 Hz), 2.59 (1H, d, J=9.5 Hz), 2.83 (1H, d, J =8.8 Hz), 3.33(1H, m), 3.57 (1H, d, J=13.2 Hz), 3.68 (1H, d, J =13.2 Hz), 4.75 (1H,brs), 7.20-7.37 (5H, m).

MS (ESI) m/z: 317 (M+H)⁺.

[α]_(D) ^(25.1)=−63.6° (c=0.129, chloroform)

Reference Example 26(+)-5-Benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane

The procedure of Reference Example 25 was repeated by using(+)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-4-one(950 mg, 2.88 mmol) to obtain crude7-amino-5-benzyl-7-methyl-5-azaspiro[2.4]heptane as a colorlesstransparent oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.37-0.45 (2H, m), 0.56-0.66 (2H, m),0.96 (3H, s), 2.48 (1H, d, J=9.0 Hz), 2.55 (1H, d, J=8.8 Hz), 2.74 (2H,d, J=9.0 Hz), 3.59 (2H, s), 7.21-7.37 (5H, m).

MS (ESI) m/z: 217 (M+H)⁺.

By using the crude 7-amino-5-benzyl-7-methyl-5-azaspiro[2.4]heptane, theprocedure of Reference Example 28 was repeated to obtain 629 mg (1.985mmol, 3 steps, 69%) of the target compound as a colorless transparentgummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.40-0.45 (1H, m), 0.50-0.55 (1H, m),0.63-0.69 (1H, m), 0.80-0.85 (1H, m), 1.20 (3H, s), 1.43 (9H, s), 2.44(1H, d, J=8.8 Hz), 2.59 (1H, d, J=9.5 Hz), 2.83 (1H, d, J =8.8 Hz), 3.33(1H, m), 3.57 (1H, d, J=13.2 Hz), 3.68 (1H, d, J =13.2 Hz), 4.75 (1H,brs), 7.20-7.37 (5H, m)

MS (ESI) m/z: 317 (M+H)⁺.

[α]_(D) ^(25.1)=+76.2° (c=0.290, chloroform)

Reference Example 27(−)-7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane

To a solution of(−)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane(581 mg, 1.836 mmol) in methanol (40 mL) was added 10% palladium-carboncatalyst (M; water content, about 50%; 349 mg), and the suspension wasstirred at room temperature for 2.5 hours in hydrogen atmosphere. Afterremoving the catalyst by filtration, the solvent was removed bydistillation under reduced pressure to obtain 434 mg (quantitative) ofthe crude target compound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.38-0.43 (1H, m), 0.55-0.60 (2H, m),0.74-0.80 (1H, m), 1.08 (3H, s), 1.44 (9H, s), 2.75 (1H, d, J=12.0 Hz),2.77 (1H, d, J=11.5 Hz), 3.13 (1H, d, J=11.5 Hz), 3.75 (1H, brd, J=12.0Hz), 4.44 (1H, brs).

MS (ESI) m/z: 227 (M+H)⁺.

[α]_(D) ^(25.1)=−63.5° (c=0.277, chloroform)

Reference Example 28(+)-7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane

To a solution of(+)-5-benzyl-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane(627 mg, 1.981 mmol) in methanol (40 mL) was added 10% palladium-carboncatalyst (M; water content, about 50%; 376 mg), and the suspension wasstirred at room temperature for 5 hours in hydrogen atmosphere. Afterremoving the catalyst, the solvent was removed by distillation underreduced pressure to obtain 452 mg (quantitative) of the crude targetcompound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.38-0.43 (1H, m), 0.55-0.60 (2H, m),0.74-0.80 (1H, m), 1.08 (3H, s), 1.44 (9H, s), 2.75 (1H, d, J=12.0 Hz),2.77 (1H, d, J=11.5 Hz), 3.13 (1H, d, J=11.5 Hz), 3.75 (1H, brd, J=12.0Hz), 4.44 (1H, brs).

MS (ESI) m/z: 227 (M+H)⁺.

[α]_(D) ^(25.1)=+59.5° (c=0.185, chloroform)

Reference Example 297-[7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

The crude(−)-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptaneproduced in Reference Example 27 (434 mg, 1.836 mmol),6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid -difluoroboron complex (663 mg, 1.836 mmol), and triethylamine(0.768 mL, 5.510 mmol) were dissolved in dimethyl sulfoxide (5 mL), andthe mixture was stirred in an oil bath at 40° C. for 14 hours. To thereaction mixture were added a mixed solution (50 mL) of ethanol andwater (ethanol : water, 4:1) and triethylamine (5 mL), and the mixturewas heated under reflux in an oil bath at 100° C. for 2 hours. Thereaction mixture was then concentrated under reduced pressure, and theresidue was dissolved in ethyl acetate (200 mL), and washed with 10%aqueous solution of citric acid (50 mL), water (50 mL×2), and saturatedaqueous solution of sodium chloride (50 mL). The organic layer was driedwith anhydrous sodium sulfate, and the solvent was removed bydistillation under reduced pressure to obtain 870 mg (1.676 mmol, 91%)of the crude target compound as a yellow foam solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.55-0.60 (1H, m), 0.68-0.73 (1H, m),0.74-0.80 (1H, m), 0.92-0.97 (1H, m), 1.22 (3H, s), 1.40 (9H, s),1.43-1.59 (2H, m), 3.13 (1H, d, J=9.8 Hz), 3.60 (3H, s), 3.75 (1H, dd,J=11.0, 3.7 Hz), 3.85 (1H, dt, J=10.2, 4.5 Hz), 4.18 (1H, d, J=10.0 Hz),4.47 (1H, m), 4.62 (1H, s), 4.79-4.99 (1H, dm), 7.83 (1H, d, J=13.7 Hz),8.68 (1H, d, J=2.7 Hz), 14.88 (0.7H, brs).

MS (ESI) m/z: 520 (M+H)⁺.

[α]_(D) ^(25.1)=128.5° (c=1.240, chloroform)

Reference Example 307-[7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

The procedure of Reference Example 29 was repeated by using the crude(+)-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptaneproduced in Reference Example 28 (452 mg, 1.981 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid -difluoroboron complex (715 mg, 1.981 mmol) to obtain 1.00 g (1.925mmol, 97%) of the crude target compound as a yellow foam solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.55-0.60 (1H, m), 0.68-0.80 (2H, m),0.91-0.97 (1H, m), 1.21 (3H, s), 1.40 (9H, s), 1.53-1.68 (2H, m), 3.04(1H, d, J=10.0 Hz), 3.61 (3H, s), 3.81 (1H, dd, J=10.7, 4.4 Hz),3.87-3.93 (1H, m), 4.24 (1H, d, J=9.8 Hz), 4.46 (1H, m), 4.65-4.85 (2H,m), 7.83 (1H, d, J=13.4 Hz), 8.76 (1H, s).

MS (ESI) m/z: 520 (M+H)⁺.

[α]_(D) ^(25.1)=+133.2° (c=2.230, chloroform)

Example 97-(7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

The7-[7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid produced in Reference Example 29 (870 mg, 1.676 mmol) was dissolvedin concentrated hydrochloric acid (10 mL) in an ice bath, and themixture was stirred at room temperature for 20 minutes and washedchloroform (20 mL×5). Saturated aqueous solution of sodium hydroxide wasadded to the aqueous layer in an ice bath to adjust the pH to 12.0, andthe pH was further adjusted to 7.4 by adding hydrochloric acid. Thesolution was extracted with a mixed solution (chloroform : methanol,10:1) (200 mL×2), and then, with lower layer of a mixed solution (200mL) (chloroform : methanol : water, 7:3:1). The organic layers werecombined and dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation. The residue was purified by recrystallizationfrom ethanol, and the crystals were dried under reduced pressure toobtain 644 mg (1.535 mmol, 92%) of the title compound as a pale pinkpowder.

mp: 195-200° C.

[α]_(D) ^(25.1)=+40.8° (c=0.147, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.49-0.56 (2H, m), 0.67-0.76 (2H, m),1.12 (3H, s), 1.43-1.64 (2H, m), 3.56 (3H, s), 3.59-3.71 (4H, m),3.99-4.04 (1H, m), 4.80-5.03 (1H, m), 7.65 (1H, d, J=13.9 Hz), 8.45 (1H,s).

Elementary analysis for C₂₁H₂₃F₂N₃O₄.0.75EtOH.0.5H₂O:

Calculated: C, 58.37; H, 6.20; F, 8.21; N, 9.08.

Found: C, 58.23; H, 5.99; F, 8.09; N, 9.02.

MS (EI) m/z: 419 (M⁺).

IR (ATR): 2964, 2843, 1726, 1612, 1572, 1537, 1452, 1439, 1387, 1360,1346, 1311, 1294, 1265, 1207 cm⁻¹.

Example 107-(7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

The procedure of Example 9 was repeated by using the7-[7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid produced in Reference Example 30 (1000 mg, 1.925 mmol) to obtain649 mg (1.546 mmol, 80%) of the title compound as a pale pink powder.

mp: 211-214° C.

[α]_(D) ^(25.1)=+128.8° (c=0.163, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.52 (2H, m), 0.73 (2H, m), 1.07 (3H,s), 1.42-1.64 (2H, m), 3.45 (1H, d, J=10.3 Hz), 3.52-3.56 (1H, m), 3.55(3H, s), 3.73 (1H, dd, J=10.0, 2.2 Hz), 3.85 (1H, d, J=9.0 Hz),3.99-4.04 (1H, m), 4.82-5.02 (1H, m), 7.64 (1H, d, J=14.4 Hz), 8.45 (1H,s).

Elementary analysis for C₂₁H₂₃F₂N₃O₄.1.0EtOH.0.5H₂O:

Calculated: C, 58.22; H, 6.37; F, 8.01; N, 8.86.

Found: C, 58.02; H, 6.13; F, 8.05; N, 9.02.

MS (EI) m/z: 419 (M⁺).

IR (ATR): 2970, 2848, 1726, 1614, 1577, 1537, 1452, 1439, 1389, 1360,1354, 1317, 1296, 1265, 1215, 1203 cm⁻¹.

Example 117-(7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid Hydrochloride

7-[7-amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid in which configuration of the amino group in the substituent atposition 7 is the same as the one produced in Example 9 (18.07 g, 39.4mmol) was suspended in methanol (54 mL), and 1N hydrochloric acid (43.4mL, 43.4 mmol) was added to the suspension at room temperature.Isopropyl alcohol (180 mL) was then added, and the mixture was stirredin a water bath at 50° C. for some time until precipitated gummymaterial thoroughly turned to crystal. After allowing to stand at roomtemperature for 1 hour, the crystals were collected by filtration, andwashed with a small amount of isopropyl alcohol (twice). The crystalswere then dried under reduced pressure to obtain the title compound12.91 g (27.2 mmol, 69%) as a yellow powder.

mp: 226-228° C.

[α]_(D) ^(25.1)=+41.1° (c=0.347, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.49-0.56 (2H, m), 0.67-0.76 (2H, m),1.12 (3H, s), 1.43-1.64 (2H, m), 3.56 (3H, s), 3.59-3.71 (4H, m),3.99-4.04 (1H, m), 4.80-5.03 (1H, m), 7.65 (1H, d, J=13.9 Hz), 8.45 (1H,s)

Elementary analysis for C₂₁H₂₃F₂N₃O₄.1HCl.1H₂O:

Calculated: C, 53.22; H, 5.53; F, 8.02; N, 8.87; Cl, 7.48.

Found: C, 53.01; H, 5.52; F, 7.90; N, 8.71; Cl, 7.53.

Example 127-(7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid hydrochloride

7-[7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid in which configuration of the tert -butoxycarbonylamino group inthe substituent at position 7 is the same as the one produced in Example9 (267.2 g) was suspended in isopropyl alcohol (1.6 L), and 6Nhydrochloric acid (405 mL, 2.43 mol) was added to the suspension whilethe suspension was stirred in an oil bath at 55° C. The mixture wasstirred at the same temperature for 3.5 hours, and after allowing tocool to room temperature, isopropyl alcohol (2.4 L) was added. Thereaction vessel was cooled in a water bath at 5° C., and the mixture wasstirred at the same temperature for 13 hours. The precipitated crystalswere collected by filtration and after air drying for several hours, thecrystals were dried under reduced pressure at 40° C. to obtain 223.3 g(471 mmol, 92%) of the target compound as a yellow powder.

Reference Example 31 Methyl3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl -carboxylate

To a solution of methyl(3R)-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate (49.5 g, 200mmol) and methyl iodide (37.4 mL, 600 mmol) in dimethylformamide (1 L),sodium hydride (in oil; content, 55%; 11.35 g: 260 mmol) was graduallyadded in 5 minutes at room temperature. After stirring at roomtemperature for 2 hours, methyl iodide (24.9 mL, 400 mmol) and sodiumhydride (in oil; content, 55%; 6.11 g; 140 mmol) were further added, andthe mixture was stirred for another 4 hours. The reaction mixture wasadded to 0.5N hydrochloric acid (1 L) in an ice bath, and extracted withethyl acetate (2 L+1 L). The organic layers were combined and washedwith water (1 L×2) and saturated aqueous solution of sodium chloride (1L), and dried with anhydrous sodium sulfate. After removing thedessicating agent by filtration, the solvent was removed by distillationunder reduced pressure. The thus obtained crude product was purified bysilica gel column chromatography (hexane : ethyl acetate,80:20→67:33→50:50→33:67) to obtain stereoisomers at position 3 of thetitle compound (stereoisomer A: 19.86 g (76.0 mmol, 38%) as a paleyellow oil, stereoisomer B: 20.22 g (77.4 mmol, 39%) as a pale yellowsolid, and a mixture of stereoisomers A and B: 9.69 g (37.1 mmol, 19%)as a pale yellow oil.

Stereoisomer A:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.21 (3H, s), 1.52 (3H, d, J=7.1 Hz),2.31 (1H, d, J=16.8 Hz), 2.73 (1H, d, J=10.0 Hz), 2.95 (1H, d, J=16.9Hz), 3.69 (1H, d, J=10.0 Hz), 3.72 (3H, s), 5.51 (1H, q, J=7.2 Hz),7.26-7.37 (5H, m).

MS (ESI) m/z: 262 (M+H)⁺.

[α]_(D) ^(25.1)=+90.1° (c=0.350, chloroform)

Stereoisomer B:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.39 (3H, s), 1.52 (3H, d, J=7.1 Hz),2.33 (1H, d, J=16.9 Hz), 2.93 (1H, d, J=16.8 Hz), 3.09 (1H, d, J=10.0Hz), 3.30 (1H, d, J=10.3 Hz), 3.61 (3H, s), 5.51 (1H, q, J=6.8 Hz),7.25-7.36 (5H, m).

MS (ESI) m/z: 262 (M+H)⁺.

[α]_(D) ^(25.1)=+120.8° (c=0.190, chloroform)

Reference Example 32 Methyl4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate

To a solution of the methyl3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate(stereoisomer A) produced in Reference Example 31 (19.86 g, 76.0 mmol)and hexamethylphosphoric triamide (30 mL) in tetrahydrofuran (300 mL), asolution of lithium diisopropyl amide in heptane, tetrahydrofuran, andethylbenzene (1.8M, 63.3 mL, 113.9 mmol) was gradually added in 15minutes at −78° C. After stirring the mixture at −78° C. for 30 minutes,ethyl iodide (12.2 mL, 152.0 mmol) was added dropwise at the sametemperature in 10 minutes. After stirring at −78° C. for 1 hour, themixture was quenched by adding saturated aqueous solution of ammoniumchloride (100 mL). The reaction mixture was extracted with ethyl acetate(300 mL), and the organic layer was washed with water (200 mL×2) andsaturated aqueous solution of sodium chloride (200 mL). After dryingwith anhydrous sodium sulfate, the dessicating agent was removed byfiltration, and the solvent was removed by distillation under reducedpressure. The resulting crude product was purified by silica gel columnchromatography (hexane : ethyl acetate, 90:10→85:15→80:20) to obtain10.63 g of the title compound (a mixture of stereoisomers of position 4,36.7 mmol, 48%) as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.01-1.07 (3.6H, m), 1.22 (2.4H, s),1.45-1.56 (3.8H, m), 1.64-1.75 (1H, m), 1.80-1.85 (0.2H, m), 2.24 (0.8H,t, J=6.7 Hz), 2.61-2.65 (1H, m), 2.81 (0.2H, t, J=7.2 Hz), 3.55 (0.2H,d, J=9.5 Hz), 3.62 (0.8H, d, J=10.0 Hz), 3.699 (2.4H, s), 3.704 (0.6H,s), 5 .50-5.56 (1H, m), 7.26-7.36 (5H, m).

MS (ESI) m/z: 290 (M+H)⁺.

Reference Example 334-Ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl -carboxylicacid

To a solution of the methyl4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate(stereoisomer A) produced in Reference Example 32 (10.63 g, 36.7 mmol)in tetrahydrofuran (330 mL) and methanol (110 mL), 2N aqueous solutionof sodium hydroxide (110 mL, 220 mmol) was added at room temperature,and the mixture was stirred in an oil bath at 60° C. for 5.5 hours. Thereaction mixture was concentrated under reduced pressure, and afteradding concentrated hydrochloric acid to the concentrate in an ice bathfor acidification, the mixture was extracted with chloroform (300mL+2×100 mL). The organic layers were combined, and dried with anhydroussodium sulfate. After removing the dessicating agent by filtration, thesolvent was removed by distillation under reduced pressure to obtain11.46 g (quantitative) of the crude title compound as a pale brownsolid. The crude product was used in the subsequent reaction with nofurther purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.03-1.08 (3.6H, m), 1.25 (2.4H, s),1.50-1.54 (3.2H, m), 1.59-1.70 (0.8H, m), 1.73-1.87 (1H, m), 2.30 (0.8H,t, J=6.6 Hz), 2.63 (0.8H, d, J=10.3 Hz), 2.67 (0.2H, d, J=9.8 Hz), 2.86(0.2H, t, J=7.2 Hz), 3.60 (0.2H, d, J=9.8 Hz), 3.69 (0.8H, d, J=10.3Hz), 5.48-5.56 (1H, m), 7.26-7.36 (5H, m).

MS (ESI) m/z: 276 (M+H)⁺.

Reference Example 34(3R*,4S*)-3-Amino-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidineand(3R*,4R*)-3-amino-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidine

To a solution of the crude4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylicacid (stereoisomer A) (11.46 g) produced in Reference Example 33 andtriethylamine (10.24 mL, 73.4 mmol) in toluene (150 mL),diphenylphosphoryl azide (10.29 mL, 47.7 mmol) was added at roomtemperature, and the mixture was stirred at room temperature for 15minutes, and in an oil bath at 90° C. for 3 hours. The reaction mixturewas diluted by adding ethyl acetate (500 mL), and the solution waswashed with saturated aqueous solution of sodium hydrogencarbonate (200mL), water (200 mL), and saturated aqueous solution of sodium chloride(200 mL) in this order. The resulting organic layer was dried withanhydrous sodium sulfate, and after removing the dessicating agent byfiltration, the solvent was removed by distillation under reducedpressure to obtain the crude product in the form of an isocyanate. Thecrude product in the form of an isocyanate was dissolved in 1,4-dioxane(80 mL), and after adding 6N hydrochloric acid (80 mL), the mixture wasstirred in an oil bath at 60° C. for 2 hours. The reaction mixture wasconcentrated under reduced pressure, and the concentrate wasazeotropically distilled with ethanol. Water (100 mL) was added to theresidue, and after stirring in an ice bath, saturated aqueous solutionof sodium hydroxide was added for alkalization. The resulting mixturewas extracted with dichloromethane (600 mL+100 mL), and the organiclayers were combined. After drying with anhydrous sodium sulfate, thedessicating agent was removed by filtration, and the solvent was removedby distillation under reduced pressure. The resulting isomer mixture wasseparated and purified by silica gel column chromatography (chloroform :methanol : triethylamine, 100:0:1→99:1:1→98:2:1) to obtain 7.00 g ofstereoisomer AA of the title compound ((3R*,4S*) configuration, 28.4mmol, 2 steps, 77%) as a pale brown gummy solid, and 1.41 g of thestereoisomer AB of the title compound ((3R*,4R*) configuration, 5.72mmol, 2 steps, 16%) as a pale brown gummy solid.

Stereoisomer AA:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.14 (3H, t, J=7.6 Hz), 1.19 (3H, s),1.43-1.55 (4H, m), 1.72-1.83 (1H, m), 2.05 (1H, t, J=6.8 Hz), 2.77 (1H,d, J=10.0 Hz), 2.99 (1H, d, J=9.8 Hz), 5.53 (1H, q, J=7.1 Hz), 7.24-7.35(5H, m).

MS (ESI) m/z: 247 (M+H)⁺.

[α]_(D) ^(25.1)=+126.6° (c=0.470, chloroform)

Stereoisomer AB:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.93 (3H, s), 1.13 (3H, t, J=7.4 Hz),1.46-1.62 (4H, m), 1.65-1.74 (1H, m), 2.11 (1H, t, J=7.1 Hz), 2.70 (1H,d, J=9.5 Hz), 3.00 (1H, d, J=9.3 Hz), 5.52 (1H, q, J=7.1 Hz), 7.24-7.35(5H, m).

MS (ESI) m/z: 247 (M+H)⁺.

[α]_(D) ²⁵ 1=+132.5° (c=0.260, chloroform)

Reference Example 35(3R*,4R*)-3-(tert-Butoxycarbonylamino)-4-ethyl-3-methyl-1-[(R)-1-phenylethyl]pyrrolidine

To a solution of(3R*,4S*)-3-amino-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidine(isomer AA, 4.16 g, 16.89 mmol) in tetrahydrofuran (100 mL) produced inReference Example 34, lithium aluminum hydride (1.282 g, 33.8 mmol) wasgradually added in 5 minutes in an ice bath, and the mixture was stirredat room temperature for 2 hours. The reaction mixture was cooled in anice bath, and after carefully adding water (1.22 mL, 67.7 mmol), 15%aqueous solution of sodium hydroxide (1.22 mL), and water (3.66 mL) inthis order, the mixture was stirred overnight at room temperature. Theinsoluble content was removed by filtration, and the residue on thefilter was washed with tetrahydrofuran (3 times), and the filtrate andthe tetrahydrofuran used for the washing were combine. The solvent wasremoved by distillation under reduced pressure, and the residue wasdissolved in dichloromethane (70 mL). Di-tert-butyl dicarbonate (5.53 g,25.3 mmol) was added, and the mixture was stirred at room temperaturefor 25 hours. The solvent was removed by distillation under reducedpressure, and the residue was purified by silica gel columnchromatography (hexane : ethyl acetate, 90:10→80:20→67:33) to obtain4.45 g (13.37 mmol, 2 steps, 79%) of the title compound as a pale brownoil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.87 (3H, t, J=7.3 Hz), 1.14-1.22 (1H,m), 1.33 (3H, d, J=6.3 Hz), 1.44 (9H, s), 1.45 (3H, s), 1.55-1.65 (1H,m), 1.74-1.82 (1H, m), 2.36 (1H, t, J=8.8 Hz), 2.63 (1H, d, J=9.5 Hz),2.71 (1H, d, J=8.1 Hz), 2.75 (1H, d, J =9.0 Hz), 3.28 (1H, q, J=6.5 Hz),4.73 (1H, brs), 7.19-7.33 (5H, m).

MS (ESI) M/Z: 333 (M+H)⁺.

[α]_(D) ^(25.1)=+5.4° (c=0.410, chloroform.)

Reference Example 36(3R*,4R*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine

To a solution of(3R*,4R*)-3-(tert-butoxycarbonylamino)-4-ethyl-3-methyl-1-[(R)-1-phenylethyl]pyrrolidine(4.43 g, 13.33 mmol) produced in Reference Example 35 in dichloromethane(40 mL), benzyl chloroformate (5.71 mL, 39.9 mmol) was added at roomtemperature, and the mixture was stirred at the same temperature for 5days. The solvent was removed by distillation under reduced pressure,and the residue was purified by silica gel column chromatography (hexane: ethyl acetate, 90:10→80:20→67:33) to obtain 3.94 g (10.86 mmol, 81%)of the title compound as a colorless transparent gummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.94-1.00 (3H, m), 1.12-1.22 (1H, m),1.41-1.47 (12H, m), 1.59-1.69 (1H, m), 1.82-1.95 (1H, m), 3.12-3.26 (2H,m), 3.65-3.75 (1H, m), 3.96 (0.3H, d, J=11.5 Hz), 4.10 (0.7H, m), 4.43(1H, brs), 5.09-5.18 (2H, m), 7.27-7.38 (5H, m).

MS (ESI) m/z: 307 (M-tBu)⁺.

[α]_(D) ^(25.1)=−13.3° (c=0.120, chloroform)

Example 137-[(3R*,4R*)-3-Amino-4-ethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1, the crude(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine(450 mg, 1.242 mmol) produced in Reference Example 36 was converted to(3R*,4R*)-3-(tert -butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine (304mg, quantitative), and 294 mg of the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (434 mg, 1.201 mmol) to obtain 282 mg (0.662mmol, 55%) of the title compound as a white powder.

mp: 90-93° C.

[α]_(D) ^(25.1)=+220.2° (c=0.113, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.96 (3H, t, J=7.4 Hz), 1.27-1.34(4H, m), 1.56-1.72 (3H, m), 1.89-1.97 (1H, m), 3.30 (1H, d, J =10.7 Hz),3.56 (3H, s), 3.61-3.70 (2H, m), 3.78 (1H, dd, J=10.6, 2.8 Hz), 4.06(1H, dd, J=13.1, 6.0 Hz), 4.82-5.01 (1H, m), 7.65 (1H, d, J=14.4 Hz),8.48 (1H, s).

Elementary analysis for C₂₁H₂₅F₂N₃O₄.0.125H₂O:

Calculated: C, 59.22; H, 6.03; F, 8.92; N, 9.87.

Found: C, 59.09; H, 5.84; F, 8.79; N, 9.89.

MS (FAB) m/z: 422 (M+H)⁺.

IR (ATR): 2958, 2873, 1724, 1618, 1541, 1508, 1431, 1363, 1313, 1277,1234 cm⁻¹.

Reference Example 37 Methyl(3R*,4R*)-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylateand methyl(3R*,4S*)-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate

The procedure of Reference Example 32 was repeated by using the methyl3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate(stereoisomer B) produced in Reference Example 31 (20.22 g, 77.4 mmol)to obtain 2.27 g of stereoisomers BA at position 4 of the title compound((3R*,4R*) configuration, 7.84 mmol, 10%) as a pale yellow oil and 7.40g of stereoisomer BB ((3R*,4S*) configuration, 25.6 mmol, 33%) as a paleyellow oil.

Stereoisomer BA:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.03 (3H, t, J=7.5 Hz), 1.25 (3H, s),1.39-1.55 (4H, m), 1.79-1.90 (1H, m), 2.78 (1H, dd, J=8.0, 6.5 Hz), 2.95(1H, d, J=9.8 Hz), 3.17 (1H, d, J=9.8 Hz), 3.64 (3H, s), 5.52 (1H, q,J=7.1 Hz), 7.25-7.35 (5H, m).

MS (ESI) m/z: 290 (M+H)⁺.

[α]_(D) ^(25.1)=+106.7° (c=0.520, chloroform)

Stereoisomer BB:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.00 (3H, t, J=7.6 Hz), 1.38-1.47 (4H,m), 1.51 (3H, d, J=7.4 Hz), 1.63-1.70 (1H, m), 2.26 (1H, dd, J=7.4, 6.4Hz), 2.98 (1H, d, J=10.3 Hz), 3.29 (1H, d, J=10.3 Hz), 3.53 (3H, s),5.52 (1H, q, J=7.1 Hz), 7.25-7.35 (5H, m).

MS (ESI) m/z: 290 (M+H)⁺.

[α]_(D) ^(25.1)+130.0° (c=0.110, chloroform)

Reference Example 38(3R*,4S*)-4-Ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylicacid

The procedure of Reference Example 33 was repeated by using the methyl(3R*,4S*)-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylate(stereoisomer BB, 2.04 g, 7.03 mmol) produced in Reference Example 37 toobtain 2.15 g (quantitative) of the crude target compound as a palebrown solid. The thus obtained crude product was used in the subsequentreaction with no further purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.98 (3H, t, J=7.6 Hz), 1.41 (3H, s),1.50-1.73 (5H, m), 2.30 (1H, dd, J=7.6, 5.9 Hz), 2.96 (1H, d, J=10.1Hz), 3.32 (1H, d, J=10.5 Hz), 5.51 (1H, q, J=7.0 Hz), 7.23-7.34 (5H, m).

MS (ESI) m/z: 276 (M+H)⁺.

Reference Example 39(3R*,4S*)-3-Amino-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidine

To a solution of the crude(3R*,4S*)-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidin-3-yl-carboxylicacid produced in Reference Example 38 (stereoisomer BB, 2.15 g, 7.03mmol) and triethylamine (1.96 mL, 14.06 mmol) in toluene (30 mL),diphenylphosphoryl azide (1.97 mL, 9.14 mmol) was added roomtemperature, and the mixture was stirred at room temperature for 15minutes, and in an oil bath at 90° C. for 3 hours. The reaction mixturewas diluted by adding ethyl acetate (200 mL), and the solution waswashed with saturated aqueous solution of sodium hydrogencarbonate (50mL), water (50 mL), and saturated aqueous solution of sodium chloride(50 mL) in this order. The resulting organic layer was dried withanhydrous sodium sulfate, and after removing the dessicating solution byfiltration, the solvent was removed by distillation under reducedpressure to obtain the crude product in the form of an isocyanate. Thethus obtained crude product in the form of an isocyanate was dissolvedin 1,4-dioxane (16 mL), and 6N hydrochloric acid (16 mL) was added tothe solution, and the mixture was stirred in an oil bath at 60° C. for3.5 hours. The reaction mixture was concentrated under reduced pressure,and the concentrate was azeotropically distilled with ethanol. To theresidue was added water (30 mL), and saturated aqueous solution ofsodium hydroxide was added in an ice bath with stirring foralkalinization. The resulting mixture was extracted with dichloromethane(150 mL+2×50 mL), and the organic layers were combined and dried withanhydrous sodium sulfate. After removing the dessicating agent byfiltration, the solvent was removed by distillation under reducedpressure to obtain 1.79 g (quantitative) of the crude target compound asa greenish brown oil. The thus obtained crude product was used in thesubsequent reaction with no further purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.13 (3H, t, J=7.6 Hz), 1.27 (3H, s),1.46-1.58 (1H, m), 1.49 (3H, d, J=7.1 Hz), 1.68-1.80 (1H, m) 2.10 (1H,t, J=7.0 Hz), 2.68 (1H, d J=9.8 Hz), 3.13 (1H, d, J =9.8 Hz), 5.51 (1H,q, J=7.2 Hz), 7.24-7.35 (5H, m). MS (ESI) m/z: 247 (M+H)⁺.

Reference Example 40(3R*,4R*)-3-(tert-Butoxycarbonylamino-4-ethyl-3-methyl-1-[(R)-1-phenylethyl]pyrrolidine

The procedure of Reference Example 35 was repeated by using the crude(3R*,4S*)-3-amino-4-ethyl-3-methyl-5-oxo-1-[(R)-1-phenylethyl]pyrrolidineproduced in Reference Example 39 (stereoisomer BB, 1.79 g, 7.03 mmol) toobtain 2.03 g (6.11 mmol, steps, 87%) of the target compound as a palered oil.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.86 (3H, t, J=8.1 Hz), 1.11-1.21 (1H,m), 1.31 (3H, d, J=6.3 Hz), 1.42 (9H, s), 1.52 (3H, s), 1.55-1.65 (1H,m), 1.78-1.86 (1H, m), 2.27 (1H, t, J=9.2 Hz), 2.55 (1H, d, J=9.5 Hz),2.81 (1H, c, J=9.5 Hz), 2.92 (1H, t, J =8.7 Hz), 3.33 (1H, q, J=6.5 Hz),4.70 (1H, brs), 7.19-7.31 (5H, m).

MS (ESI) m/z: 333 (M+H)⁺.

[α]_(D) ^(25.1)=+30.3° (c=0.405, chloroform)

Reference Example 41(3R*,4R*)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine

The procedure of Reference Example 36 was repeated by using the(3R*,4R*)-3-(tert-butoxycarbonylamino)-4-ethyl-3-methyl-1-[(R)-1-phenylethyl]pyrrolidineproduced in Reference Example 40 (stereoisomer BB, 2.03 g, 6.11 mmol) toobtain 1.752 g (4.83 mmol, 79%) of the target compound as a pale pinkgummy solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.94-1.00 (3H, m), 1.12-1.22 (1H, m),1.41-1.47 (12H, m), 1.59-1.69 (1H, m), 1.82-1.95 (1H, m), 3.12-3.26 (2H,m), 3.65-3.75 (1H, m), 3.96 (0.3H, d, J=11.5 Hz), 4.10 (0.7H, m), 4.43(1H, brs), 5.09-5.18 (2H, m), 7.27-7.38 (5H, m).

MS (ESI) m/z: 307 (M-tBu)⁺.

[α]_(D) ^(25.1)=+10.5° (c=0.260, chloroform)

Example 147-[(3R*,4R*)-3-Amino-4-ethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

By using a procedure similar to Example 1, the(3R*,4R*)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidineproduced in Reference Example 41 (439 mg, 1.211 mmol) was converted tocrude (3R*,4R*)-3-(tert-butoxycarbonylamino)-4-ethyl-3-methylpyrrolidine (285 mg,quantitative), and 283 mg of the product was reacted with6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (434 mg, 1.201 mmol) to obtain 256 mg (0.565mmol, 47%) of the title compound as a white powder.

mp: 167-169° C.

[α]_(D) ^(25.1)=97.6° (c=0.127, 0.1N NaOH)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.96 (3H, t, J=6.9 Hz), 1.25-1.44(5H, m), 1.49-1.68 (2H, m), 1.94 (1H, m), 3.37 (1H, d, J=10.5 Hz),3.54-3.76 (6H, m), 3.97-4.02 (1H, m), 5.02 (1H, dm, J=66.4 Hz), 7.65(1H, d, J=14.6 Hz), 8.39 (1H, s)

Elementary analysis for C₂₁H₂₅F₂N₃O₄.0.5H₂O.0.5EtOH:

Calculated: C, 58.27; H, 6.45; F, 8.38; N, 9.27.

Found: C, 58.31; H, 6.46; F, 8.23; N, 9.08.

MS (FAB) m/z: 422 (M+H)⁺.

IR (ATR): 2967, 2939, 2883, 2831, 1728, 1612, 1577, 1537, 1493, 1456,1439, 1389, 1358, 1302, 1284, 1261 cm⁻¹.

Reference Example 42 tert-Butyl2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-4-carboxylate

To a suspension of 2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-4-carboxylicacid (1165 g, 4.994 mol) in dichloromethane (10 L) was addedO-tert-butyl-N,N′-diisopropylurea (3020 g, 15.00 mol) at roomtemperature with stirring, and the reaction system was cooled by addingice water to outer bath when increase of inner temperature and start ofthe refluxing were noted. When the temperature decreased to roomtemperature, the reaction mixture was stirred for 1 hour after removingthe ice bath, and for 3 hours by heating to 40° C. After stirring thereaction mixture in an ice bath for 1 hour, the insoluble content wasseparated by filtration, and the solvent was removed by distillationunder reduced pressure. The residue was purified by silica gel columnchromatography (silica gel, 4 kg; elution solution, hexane : ethylacetate, 3:1) to obtain 925.2 g (64%) of a mixture of isomers atposition 4 as a pale yellow syrup. Although separation of the isomerswas easy, the isomers were used without separation since the subsequentstep involved racemization. ¹H-NMR spectrum of the isomers authenticsample is shown below.

Low Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.45 (9H, s), 1.54 (3H, d, J=7.08),2.59-2.74 (2H, m), 2.95-3.03 (1H, m), 3.14 (1H, dd, J=9.77, 8.79 Hz),3.49 (1H, dd, J=9.77, 6.35 Hz), 7.26-7.36 (5H, m).

High Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.36 (9H, s), 1.53 (3H, d, J=7.32),2.59-2.75 (2H, m), 3.02-3.11 (1H, m), 3.16 (1H, dd, J=10.01, 5.62 Hz),3.51 (1H, dd, J=10.01, 8.54 Hz), 7.24-7.36 (5H, m)

Reference Example 43 tert-Butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-4-carboxylate (30.05 g, 0.104mol) in N,N′-dimethylformamide (210 mL), iodomethane 26.0 mL (59.28 g,0.418 mol), and then sodium hydride 155%, in oil, 11.35 g, 0.260 mol)was added at room temperature in nitrogen atmosphere while stirring themixture. When inner temperature increased to about 50° C., the reactionmixture was cooled to 30° C. by adding ice water to the outer bath.After changing the bath to a water bath at an outer temperature of 17°C., the mixture was stirred for 23 hours. The reaction mixture waspoured into cold aqueous solution of citric acid (1 L of 10% citric acidand 500 g of ice), and after stirring the mixture for 30 minutes, themixture was extracted with ethyl acetate (800 mL, 500 mL). The organiclayers were combined, and washed with saturated aqueous solution ofsodium chloride. After drying with anhydrous sodium sulfate, the mixturewas subjected to filtration, and the filtrate was concentrated underreduced pressure. The concentrate was purified by flash silica gelcolumn chromatography (elution was started at hexane ethyl acetate of5:1, and after elution of the low polarity isomer, hexane : ethylacetate was changed to 4:1) to obtain 10.63 g (33.7%) of high polarityisomer the title compound as a white solid. 14.91 g (47.3%) of lowpolarity isomer of tert-butyl(4R)-4-methyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-4-carboxylate wasalso obtained.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (12H, s), 1.52 (3H, d, J=7.10 Hz),2.27 (1H, d, J=17.0 Hz), 2.93 (1H, d, J=17.0 Hz), 3.05 (1H, d, J=10.1Hz), 3.32 (1H, d, J=10.1 Hz), 5.50 (1H, q, J=7.1 Hz), 7.23-7.38 (5H, m).

Reference Example 44 tert-Butyl(3R)-4-hydroxy-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(10.0 g, 33.0 mmol) and triethyl phosphite (6.78 mL, 39.6 mmol) inanhydrous tetrahydrofuran (165 mL), lithium bistrimethylsilylamide (46.1mL, 46.1 mmol, 1.0M solution in tetrahydrofuran) was added at −5° C.,and the mixture was stirred at the same temperature for 30 minutes.After bubbling oxygen gas into the reaction mixture for 2 hours,saturated aqueous solution of ammonium chloride (150 mL) was added tothe mixture in an ice bath, and the mixture was concentrated underreduced pressure. Water (100 mL) was added to the residue and themixture was extracted with ethyl acetate (200 mL×2). The organic layerwas washed with saturated aqueous solution of sodium chloride (200 mL),and dried with anhydrous sodium sulfate. After removing the solvent bydistillation under reduced pressure, the residue was purified by silicagel column chromatography (hexane : ethyl acetate=1:1→1:4) to obtain9.61 g (91.3%) of the title compound as a pale yellow oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.24-1.54 (15H, m), 2.64-4.54 (3H, m),5.44-5.51 (1H, m), 7.26-7.37 (5H, m)

MS (FAB⁺) m/z: 320 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₈H₂₆NO₄: δ 320.1862; Found: 320.1853.

IR (ATR) ν: 3363, 2978, 2935, 2360, 1716, 1684, 1489, 1456, 1369, 1304,1269, 1230, 1167 cm⁻¹.

Reference Example 45 tert-Butyl(3R)-4-hydroxy-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl(3R)-4-hydroxy-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(9.35 g, 29.3 mmol) in tetrahydrofuran (150 mL), a solution of borane intetrahydrofuran (82.6 mL, 96.6 mmol, 1.17M solution in tetrahydrofuran)was added in an ice bath, and the mixture was stirred room temperaturefor 14 hours. Water (20 mL), ethanol (80 mL), and triethylamine (20 mL)were added to the reaction mixture in an ice bath, and the mixture washeated under reflux in an oil bath at 88° C. for 2 hours. Afterconcentrating the reaction mixture under reduced pressure, water (200mL) was added and the mixture was extracted with ethyl acetate (200mL×2). The organic layer was washed with saturated aqueous solution ofsodium chloride (200 mL), and dried with anhydrous sodium sulfate. Thesolvent was removed by distillation under reduced pressure, and theresidue was purified by silica gel column chromatography (hexane : ethylacetate=3:1→1:2) to obtain 4.75 g (53.1%) of the title compound as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.25 (3H, s), 1.32 (3H, d, J=6.6 Hz),1.44 (9H, s), 1.92 (1H, d, J=6.6 Hz), 2.39 (1H, d, J=9.5 Hz), 2.69 (1H,dd, J=9.8, 3.9 Hz), 2.75-2.82 (1H, m), 2.92 (1H, d, J=9.8 Hz), 3.31 (1H,q, J=6.6 Hz), 4.38-4.45 (1H, m), 7.20-7.30 (5H, m).

MS (FAB⁺) m/z: 306 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₈H₂₈NO₃: 306.2069; Found: 306.2064.

IR (ATR) ν: 3450, 2976, 2931, 2785, 2359, 1790, 1720, 1603, 1493, 1477,1454, 1367, 1281, 1255, 1211 cm⁻¹.

Reference Example 46 tert-Butyl(3R)-1-benzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate

To a solution of tert-butyl(3R)-4-hydroxy-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(2.00 g, 6.55 mmol) in ethanol (10 mL), 1N hydrochloric acid (6.88 mL,6.88 mmol) was added at room temperature, and the mixture was stirredfor 10 minutes. After concentrating the reaction mixture under reducedpressure and dissolving the residue in ethanol (50 mL), 10%palladium-carbon catalyst (200 mg) was added, and the suspension wasstirred in an oil bath at 50° C. for 14 hours in hydrogen atmosphere.After filtering the reaction mixture, the filtrate was concentrated, andthe diethylether (30 mL) and saturated sodium hydrogencarbonate (30 mL)were added to the residue, and benzyloxycarbonyl chloride (982 μl, 6.88mmol) was added in an ice bath. The mixture was stirred at roomtemperature for 3 hours, and the reaction mixture was extracted withethyl acetate (100 mL×2). The organic layer was washed with saturatedaqueous solution of sodium chloride (20 mL), and dried with anhydroussodium sulfate. The solvent was removed by distillation under reducedpressure and the residue was purified by silica gel columnchromatography (hexane : ethyl acetate=2:1→1:2) to obtain 2.00 g (91.1%)of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.30 (3H, s), 1.44 (9H, s), 2.10 (1H, dd,J=30.9, 3.8 Hz), 3.26-3.45 (2H, m), 3.68-3.79 (2H, m), 4.45-4.51 (1H,m), 5.13 (2H, s), 7.28-7.38 (5H, m)

MS (FAB⁺) m/z: 336 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₈H₂₆NO₅: 336.1811; Found: 336.1789.

IR (ATR) ν: 3421, 2978, 2941, 2885, 2364, 1788, 1707, 1687, 1498, 1456,1423, 1367, 1319, 1257, 1213, 1161 cm⁻¹.

Reference Example 47 tert-Butyl(R)-1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate

To a solution of oxalyl chloride (1.01 mL, 11.7 mmol) in dichloromethane(40 mL), a solution of dimethyl sulfoxide (1.11 mL, 15.6 mmol) indichloromethane (5 mL) was added at −78° C., and the mixture was stirredfor 10 minutes. After adding a solution of tert-butyl(R)-1-benzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate(1.97 g, 5.87 mmol) in dichloromethane (15 mL) and stirring the mixturefor 1 hour, triethylamine (5.98 mL, 42.9 mmol) was added, and themixture was stirred −78° C. for 30 minutes and in an ice bath for 30minutes. To the reaction mixture were added saturated aqueous solutionof ammonium chloride (50 mL) and water (100 mL), and the mixture wasextracted with ethyl acetate (200 mL×2). The organic layer was washedwith water (100 mL) and saturated aqueous solution of sodium chloride(100 mL), and dried with anhydrous sodium sulfate. The solvent wasremoved by distillation under reduced pressure, and the residue waspurified by silica gel column chromatography (hexane-ethylacetate=10:1→3:2) to obtain 1.68 g (85.8%) of the title compound as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (3H, d, J=4.9 Hz), 1.38 (9H, s),3.46 (1H, d, J=12.0 Hz), 3.81 (1H, d, J=19.0 Hz), 4.10-4.19 (1H, m),4.35 (1H, dd, J=5.9, 12.0 Hz), 5.19 (2H, s), 7.30-7.40 (5H, m).

MS (FAB⁺) m/z: 334 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₈H₂₄NO₅: 334.1654; Found: 334.1643.

IR (ATR) ν: 2981, 2941, 2889, 1768, 1711, 1498, 1454, 1421, 1369, 1290,1269, 1196, 1134 cm⁻¹.

Reference Example 48 tert-Butyl(S)-1-benzyloxycarbonyl-3-methyl-4-methylene pyrrolidine-3-carboxylate

To a solution of methyltriphenylphosphonium bromide (2.02 g, 5.65 mmol)in anhydrous tetrahydrofuran (30 mL), n-butyl lithium (3.11 mL, 4.78mmol, 1.54M solution in hexane) was added at −78° C., and the mixturewas stirred for 20 minutes. At the same temperature, a solution oftert-butyl(R)-1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate (1.45 g,4.35 mmol) in anhydrous tetrahydrofuran (15 mL) was added, and aftergradually increasing the temperature, the mixture was stirred at 55° C.for 3 hours. To the reaction mixture was added 10% aqueous solution ofcitric acid (30 mL) in an ice bath, and the solution was concentratedunder reduced pressure. Water (100 mL) was added to the concentrate, andthe mixture was extracted with ethyl acetate (100 mL×2). The organiclayer was washed with saturated aqueous solution of sodiumhydrogencarbonate (50 mL) and saturated aqueous solution of sodiumchloride (50 mL), and dried with anhydrous sodium sulfate. The solventwas removed by distillation under reduced pressure to obtain 710 mg(49.3%) of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.36 (3H, d, J=6.6 Hz), 1.39 (9H, d,J=2.0 Hz), 3.24 (1H, dd, J=13.7, 11.2 Hz), 4.05-4.16 (2H, m), 4.19-4.29(1H, m), 5.04-5.18 (4H, m), 7.29-7.38 (5H, m)

MS (FAB⁺) m/z: 332 (M+H)⁺.

HRMS (FAB⁺) m/z: Calc for C₁₉H₂₆NO₄: 332.1862; Found: 332.1869.

IR (ATR)ν: 2976, 2935, 2873, 1705, 1498, 1450, 1417, 1362, 1308, 1275,1257, 1213, 1159 cm⁻¹.

Reference Example 49 (S)-1-Benzyloxycarbonyl-3-methyl-4-methylenepyrrolidine-3-carboxylic Acid

To a solution of tert-butyl (S)-1-benzyloxycarbonyl-3-methyl-4-methylenepyrrolidine-3-carboxylate (710 mg, 2.14 mmol) in dichloromethane (8 mL),trifluoroacetic acid (4 mL) was added in an ice bath, and the mixturewas stirred at room temperature for 2 hours. Saturated aqueous solutionof sodium hydrogencarbonate (20 mL) was added to the reaction mixture,and the mixture was washed with diethylether (20 mL). After adding 1Nhydrochloric acid to the aqueous layer, the mixture was extracted withchloroform (100 mL×2), and the organic layer was dried with anhydroussodium sulfate. The solvent was removed by distillation under reducedpressure to obtain 590 mg (100%) of the title compound as a colorlessoily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.44 (3H, s), 3.23-3.36 (1H, m),4.11-4.30 (3H, m), 5.09-5.25 (4H, m), 7.27-7.39 (5H, m).

MS (FAB⁺) m/z: 276 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₅H₁₈NO₄: 276.1236, Found: 276.1222

IR (ATR) ν: 3064, 3032, 2979, 2945, 2877, 2362, 1705, 1672, 1498, 1423,1362, 1309, 1257, 1213, 1167, 1124 cm⁻¹.

Reference Example 50(S)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3-methyl-4-methylenePyrrolidine

To a solution of (S)-1-benzyloxycarbonyl-3-methyl-4-methylenepyrrolidine-3-carboxylic acid (590 mg, 2.14 mmol) in toluene (21 mL),triethylamine (597 μl, 4.29 mmol) and diphenyl phosphoryl azide (508 μl,2.36 mmol) was added, and the mixture was stirred at room temperaturefor 1 hour. The mixture was then heated under reflux in an oil bath at125° C. for 1 hour, and concentrated. The concentrate was dissolved in1,4-dioxane (8 mL) and 6N hydrochloric acid (4 mL) was added. Afterstirring for 1 hour, water (20 mL) was added to the reaction mixture,and the mixture was washed with diethylether (50 mL). The aqueous layerwas alkalized with saturated aqueous solution of sodiumhydrogencarbonate, and extracted with chloroform (100 mL×2). The organiclayer was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The residue wasdissolved in dichloromethane (8 mL), and di-tert-butyl dicarbonate (936mg, 4.29 mmol) was added in an ice bath. The solvent was stirred at 25°C. for 19 hours, and the reaction mixture was concentrated under reducedpressure. The concentrate was purified by silica gel columnchromatography (hexane:ethyl acetate=5:1→3:1) to obtain 514 mg (69.2%)of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.42 (3H, s), 1.46 (3H, s), 3.56 (1H, t,J=9.2 Hz), 3.85-3.98 (1H, m), 4.13-4.26 (2H, m), 4.62-4.74 (1H, m),5.01-5.17 (4H, m), 7.27-7.38 (5H, m).

MS (FAB⁺) m/z: 347 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₉H₂₇N₂O₄: 347.1971, Found: 347.1954

IR (ATR) ν: 3334, 2976, 2931, 2873, 1695, 1498, 1448, 1419, 1390, 1363,1282, 1244, 1215, 1165 cm⁻¹.

Reference Example 51(S)-3-(tert-Butoxycarbonylamino)-3-methyl-4-methylene Pyrrolidine

Ammonia gas was bubbled into a solution of(S)-1-benzyloxycarbonyl-3-(tert-butoxycarborylamino)-3-methyl-4-methylenepyrrolidine (488 mg, 1.41 mmol) in tetrahydrofuran (7 mL) at −78° C. toproduce a mixed solution of liquid ammonia and tetrahydrofuran (20 mL),and sodium (162 mg, 7.04 mmol) was added. The mixture was stirred at thesame temperature for 10 minutes. Saturated aqueous solution of ammoniumchloride (20 mL) was added at −78° C., and the mixture was stirred atroom temperature for 30 minutes. To the reaction mixture was addedsaturated aqueous solution of sodium hydrogencarbonate (50 mL), and thesolution was extracted with chloroform (200 mL×2). The organic layer wasdried with anhydrous sodium sulfate, and the solvent was removed bydistillation under reduced pressure to obtain 218 mg (72.9%) of thetitle compound as a colorless crystals.

[α]_(D) ^(25.1)=−74.09° (c=1.04, CHCl₃)

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.41-1.416 (12H, m), 2.90 (1H, d, J=11.7Hz), 3.45 (1H, d, J=14.2 Hz), 3.50-3.57 (1H, m), 3.69 (1H, d, J=16.1Hz), 4.64 (1H, brs), 5.01 (1H, t, J=2.2 Hz), 5.04 (1H, t, J=2.2 Hz).

MS (FAB⁺) m/z: 213 (M+H)⁺.

HRMS (FAB⁺) m/z: Calcd for C₁₁H₂₁N₂O₂: 213.1603; Found: 213.1600.

IR (ATR) ν: 3284, 3199, 2978, 2922, 28329, 1695, 1660, 1556, 1441, 1365,1333, 1288, 1279, 1248, 1176 cm⁻¹ .

Example 15 7-[(3S)-3-Amino-3-methyl-4-methylenepyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

To a solution of (3S)-3-(tert-butoxycarbonylamino)-3-methyl-4-methylenepyrrolidine (218 mg, 1.03 mmol) in dimethyl sulfoxide (3.1 mL),triethylamine (156 μl, 1.12 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroborane complex (337 mg, 934 μmol) were added, and themixture was stirred at room temperature for 3 days. The reaction mixturewas concentrated, and to the concentrate were added a mixed solution ofethanol and water (ethanol:water, 4:1) (25 mL) and triethylamine (5 mL),and the mixture was heated under reflux in an oil bath at 90° C. for 3hours. The reaction mixture was concentrated under reduced pressure, and10% aqueous solution of citric acid (50 mL) and water (50 mL) were addedto the residue, and the mixture was extracted with ethyl acetate (100mL×2). The organic layer was washed with water (50 mL) and saturatedaqueous solution of sodium chloride (50 mL). The organic layer was driedwith anhydrous sodium sulfate, and the solvent was removed bydistillation under reduced pressure. The residue was dissolved inconcentrated hydrochloric acid (10 mL) in an ice bath, and the solutionwas stirred at room temperature for 10 minutes. Water (50 mL) was addedto the reaction mixture, and the mixture was washed with chloroform (50mL×2). In an ice bath, 10 mol/l aqueous solution of sodium hydroxide wasadded to the aqueous layer to pH 12.0, and the pH was adjusted to 7.4 byadding concentrated hydrochloric acid. The solution was extracted withchloroform (100 mL×2). The organic layer was dried with anhydrous sodiumsulfate, and the solvent was removed by distillation under reducedpressure. The residue was purified by recrystallization from ethanol,and the crystals were dried under reduced pressure to obtain 224 mg(57.9%) of the title compound as a pale yellow crystals.

mp: 100-102° C.

[α]_(D) ^(25.1)=118.9° (c=0.39, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.40 (3H, s), 1.49-1.72 (2H, m)3.51-3.67 (5H, m), 4.00-4.09 (1H, m), 4.46-4.53 (1H, m), 4.21 (1H, d,J=14.9 Hz), 4.97 (1H, d, J=64.5 Hz), 5.07 (1H, s), 5.18 (1H, s), 7.71(1H, d, J=14.4 Hz), 8.47 (1H, d, J=1.2 Hz).

Elementary analysis for C₂₀H₂₁F₂N₃O₄.0.5E₂O:

Calculated: C, 57.97; H, 5.35; F, 9.17; N, 10.14.

Found: C, 57.91; H, 5.42; F, 9.41; M, 10.15.

MS (ESI) m/z: 406 (M+H)⁺.

IR (ATR) ν: 2839, 1724, 1614, 1577, 1541, 1508, 1435, 1396, 1356, 1338,1323, 1271, 1188 cm⁻¹.

Reference Example 52(3S)-10-[7-(tert-Butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane-5-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid

(−)-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane (391mg, 1.73 mmol),(3S)-9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1.2.3-de][1,4]benzoxazine-6-carboxylicacid—difluoroborane complex (580 mg, 1.76 mmol), and triethylamine(0.490 mL, 3.52 mmol) were dissolved in dimethyl sulfoxide (5 mL), andthe solution was stirred in an oil bath at 40° C. for 24 hours. To thereaction mixture were added a mixed solution of ethanol and water(ethanol:water, 5:2) (7 mL) and triethylamine (2 mL), and the mixturewas heated under reflux in an oil bath at 100° C. for 3.5 hours. Thereaction mixture was concentrated under reduced pressure, and theconcentrate was dissolved in ethyl acetate. The solution was washed with10% aqueous solution of citric acid, water, and saturated aqueoussolution of sodium chloride. The organic layer was dried with anhydroussodium sulfate, and the solvent was removed by distillation underreduced pressure. The residue was purified by silica gel columnchromatography (dichloromethane:methanol, 98:2) to obtain 761 mg (5.37mmol, 90%) of the title compound as a yellow oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.53-0.60 (1H, m), 0.63-0.71 (1H, m),0.71-0.79 (1H, m), 0.87-0.96 (1H, m), 1.21 (3H, s), 1.42 (9H, s), 1.61(3H, d, J=6.8 Hz), 3.32 (1H, dd, J=10.3, 2.2 Hz), 3.89 (1H, dd, J=11.0,2.2 Hz), 4.15-4.69 (5H, m), 7.68 (1H, d, J=14.2 Hz), 8.54 (1H, s).

MS (ESI) m/z: 488 (M+H)⁺.

Example 16(3S)-10-(7-Amino-7-methyl-5-azaspiro[2.4]heptane-5-yl)-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicAcid

(3S)-10-[7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane-5-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylicacid (761 mg, 1.56 mmol) was dissolved in concentrated hydrochloric acid(6.5 mL) in an ice bath, and the mixture was stirred at room temperaturefor 120 minutes. The reaction mixture was washed with chloroform, andsaturated aqueous solution of sodium hydroxide was added to the aqueouslayer in an ice bath to a pH of 12.0. Hydrochloric acid was then addedto adjust pH to 7.4, end the solution was extracted with lower layer ofa mixed solution (chloroform:methanol water, 7:3:1). After combining theorganic layers, and drying with anhydrous sodium sulfate, the solventwas removed by distillation under reduced pressure. Chloroform was addedto the residue, and the insoluble content was removed by filtration. Thefiltrate distilled under reduced pressure. The residue was purified bycrystallization from ethanol, and dried under reduced pressure to obtain260 mg (0.68 mmol, 43%) of the title compound as a yellow powder.

mp: 268-270° C.

[α]_(D) ²⁵=−114° (c=0.200, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.52-0.58 (2H, m), 0.70-0.76 (2H, m),1.08 (3H, s), 1.49 (3H, d, J=6.8 Hz), 3.55-3.68 (2H, m), 3.73-3.85 (2H,m), 4.28 (1H, d, J=9.3 Hz), 4.42-4.48 (1H, m), 4.52-4.61 (1H, m), 7.48(1H, d, J=14.4 Hz), 8.29 (1H, s).

Elementary analysis for C₂₀H₂₂FN₃O₄:

Calculated: C, 62.01; H, 5.72; F, 4.90; N, 10.85.

Found: C, 62.00; H, 5.65; F, 4.85; N, 10.69.

MS (FAB) m/z: 388 (M+H)⁺.

IR (ATR) ν: 3365, 2979, 2877, 1619, 1572, 1519, 1444, 1413, 1398, 1376,1359, 1338, 1328, 1307, 1280, 1108, 1081, 867 cm⁻¹.

Reference Example 53 tert-Butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate andtert-butyl(3R)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-4-carboxylate (30.05 g, 0.104mol) in N,N-dimethylformamide (210 mL), iodomethane (26.0 mL, 59.28 g,0.418 mol), and then, sodium hydride (55%, in oil, 11.35 g, 0.260 mol)was added at room temperature in nitrogen atmosphere while stirring themixture. When inner temperature increased to about 50° C., the reactionmixture was cooled to 30° C. by adding ice water to the outer bath.After changing the bath to a water bath at an outer temperature of 17°C., the mixture was stirred for 23 hours. The reaction mixture waspoured into cold aqueous solution of citric acid (1 L of 10% citric acidand 500 g of ice), and after stirring the mixture for 30 minutes, themixture was extracted with ethyl acetate (800 mL, 500 mL). The organiclayers were combined, and washed with saturated aqueous solution ofsodium chloride. After drying with anhydrous sodium sulfate, the mixturewas subjected to filtration, and the filtrate was concentrated underreduced pressure. The concentrate was purified by flash silica gelcolumn chromatography (elution was started at hexane ethyl acetate of5:1, and after elution of the low polarity isomer, hexane:ethyl acetatewas changed to 4:1) to obtain 10.63 g (33.7%) of high polarity isomer oftert-butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate as awhite solid. 14.91 g (47.3%) of low polarity isomer of tert-butyl(3R)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate wasalso obtained.

High Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (9H, s), 1.52 (3H, d, J=7.10 Hz),2.27 (1H, d, J=17.0 Hz), 2.93 (1H, d, J=17.0 Hz), 3.05 (1H, d, J=10.1Hz), 3.32 (1H, d, J=10.1 Hz), 5.50 (1H, q, J=7.1 Hz), 7.23-7.38 (5H, m).

Low Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.18 (3H, s), 1.44 (9H, s), 1.52 (3H, d,J=7.1 Hz), 2.26 (1H, d, J=16.9 Hz), 2.63-2.69 (1H, m), 2.91 (1H, d,J=16.9 Hz), 3.63 (1H, d, J=10.0 Hz), 5.51 (1H, q, J=7.1 Hz), 7.26-7.37(5H, m).

Reference Example 54 tert-Butyl(3S)-4-hydroxy-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl(3R)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(9.90 g, 32.6 mmol) and tri ethyl phosphite (6.71 mL, 39.1 mmol) inanhydrous tetrahydrofuran (165 mL), lithium bistrimethylsilyl amide(45.7 mL, 45.7 mmol, 1.0M solution in tetrahydrofuran) was added at −5°C., and the mixture was stirred at the same temperature for 30 minutes.After bubbling oxygen gas into the reaction mixture for 30 minutes,saturated aqueous solution of ammonium chloride (150 mL) was added in anice bath, and the mixture was concentrated under reduced pressure. Water(100 mL) was added to the residue and the mixture was extracted withethyl acetate (200 mL×2). The organic layer was washed with saturatedaqueous solution of sodium chloride (200 mL), and dried with anhydroussodium sulfate. After the filtration, the filtrate was concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (hexane ethyl acetate, 1:1→1:4) to obtain 7.73 g(74%) of the title compound as a pale yellow oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.24-1.56 (15H, m), 2.61-2.73 (1H, m),3.43-3.57 (1H, m), 4.02-4.15 (2H, m), 5.45-5.54 (1H, m), 7.26-7.38 (5H,m).

Reference Example 55 tert-Butyl(3S)-4-hydroxy-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl(3S)-4-hydroxy-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(5.13 g, 16.1 mmol) in tetrahydrofuran (100 mL), 1.17M solution ofborane in tetrahydrofuran (45.3 mL, 53.1 mmol) was added in an ice bath,and the mixture was stirred at room temperature for 13 hours. Afterconcentrating the reaction mixture under reduced pressure, water (10mL), ethanol (100 mL), and triethylamine (5 mL) were added to theconcentrate, and the mixture was heated under reflux in an oil bath at90° C. for 2 hours. The reaction mixture was concentrated under reducedpressure, and water (200 mL) was added to the concentrate. The mixturewas extracted with ethyl acetate (200 mL×2). The resulting organic layerwas washed with saturated aqueous solution of sodium chloride (200 mL),and dried with anhydrous sodium sulfate. After the filtration, thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (hexane:ethyl acetate,3:1→1:2) to obtain 1.50 g (31%) of the title compound as a colorlessoily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.26 (3H, s), 1.33 (3H, d, J=6.6 Hz),1.44 (9H, s), 2.42 (1H, dd, J=3.8, 3.9 Hz), 2.49 (1H, d, J=10.0 Hz),2.94 (2H, dd, J=10.0, 5.9 Hz), 3.30 (1H, q, J=6.6 Hz), 4.45 (1H, br s),7.20-7.30 (5H, m)

MS (ESI) m/z: 306 (M+H)⁺.

Reference Example 56 tert-Butyl(3S)-1-benzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate

To a solution of tert-butyl(3S)-4-hydroxy-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(1.49 g, 4.88 mmol) in ethanol (30 mL), 1N hydrochloric acid (5.12 mL,5.12 mmol) was added at room temperature, and the mixture was stirred at10 minutes. 10% palladium-carbon catalyst (1.40 g) was added to thereaction mixture, and the suspension was stirred in an oil bath at 40°C. for 2 hours in hydrogen atmosphere at normal pressure. Afterfiltering the reaction mixture and concentrating the filtrate,tetrahydrofuran (20 mL), water (20 mL), and sodium hydrogencarbonate(2.05 g, 24.4 mmol) were added to the residue, and benzyloxycarbonylchloride (836 μl, 5.86 mmol) was further added in an ice bath. Thereaction mixture was stirred at room temperature for 2 hours, andextracted with ethyl acetate (100 mL×2). The resulting organic layer waswashed with saturated aqueous solution of sodium chloride (20 mL), anddried with anhydrous sodium sulfate. After the filtration, the filtratewas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (hexane:ethyl acetate, 2:1→1:2) toobtain 1.48 g (90%) of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.30 (3H, s), 1.44 (9H, s), 2.10 (1H, dd,J=30.9, 3.8 Hz), 3.26-3.45 (2H, m), 3.68-3.79 (2H, m), 4.45-4.51 (1H,m), 5.13 (2H, s), 7.28-7.38 (5H, m).

MS (ESI) m/z: 358 (M+Na)⁺.

Reference Example 57 tert-Butyl(3S)-1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate

To a solution of oxalyl chloride (736 μl, 8.58 mmol) in dichloromethane(30 mL), a solution of dimethyl sulfoxide (811 μl, 11.4 mmol) indichloromethane (5 mL) was added at −78° C., and the mixture was stirredfor 10 minutes. A solution of (3S)-1-tert-butyl tert-butylbenzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate (1.44 g,4.29 mmol) in dichloromethane (10 mL) was added, and the mixture wasstirred for 1 hour. Triethylamine (4.37 mL, 31.4 mmol) was then added,and the mixture was stirred at −78° C. for 30 minutes, and in an icebath, for 30 minutes. To reaction mixture were added saturated aqueoussolution of ammonium chloride (50 mL) and water (100 mL), and themixture was extracted with ethyl acetate (200 mL×2). The organic layerwas washed with water (100 mL) and saturated aqueous solution of sodiumchloride (100 mL), and dried with anhydrous sodium sulfate. After thefiltration, the filtrate was concentrated under reduced pressure bysilica gel column chromatography (hexane-ethyl acetate, 10:1→3:2) toobtain 1.37 g (96%) of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (3H, d, J=4.9 Hz), 1.38 (9H, s),3.46 (1H, d, J=12.0 Hz), 3.81 (1H, d, J=19.0 Hz), 4.10-4.19 (1H, m),4.35 (1H, dd, J=5.9, 12.0 Hz), 5.19 (2H, s), 7.30-7.40 (5H, m).

Reference Example 58 tert-Butyl(3R)-1-benzyloxycarbonyl-3-methyl-4-methylene pyrrolidine-3-carboxylate

To a solution of methyltriphenylphosphonium bromide (1.67 g, 4.68 mmol)in anhydrous tetrahydrofuran (20 mL), n-butyl lithium (2.54 mL, 3.96mmol, 1.56M solution in hexane) was added at −78° C., and the mixturewas stirred for 20 minutes. A solution of tert-butyl(3S)-1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate (1.20g, 3.60 mmol) in anhydrous tetrahydrofuran (4 mL) was added at the sametemperature, and the temperature was gradually increased, and themixture was stirred at 55° C. for 3 hours. To the reaction mixture wasadded 10% aqueous solution of citric acid (50 mL) in an ice bath, andthe mixture was concentrated under reduced pressure. Water (100 mL) wasadded to the concentrate, and the mixture was extracted with ethylacetate (100 mL×2). The organic layer was washed with saturated aqueoussolution of sodium chloride (50 mL), and dried with anhydrous sodiumsulfate. After the filtration, the filtrate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (hexane-ethyl acetate, 10:1→5:1) to obtain 750 mg (63%)of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.36 (3E, d, J=6.6 Hz), 1.39 (9H, d,J=2.0 Hz), 3.24 (1H, dd, J=13.7, 11.2 Hz), 4.05-4.16 (2H, m), 4.19-4.29(1H, m), 5.04-5.18 (4H, m), 7.29-7.38 (5H, m).

Reference Example 59 (3R)-1-Benzyloxycarbonyl-3-methyl-4-methylenepyrrolidine-3-carboxylic Acid

To a solution of tert-butyl(3R)-1-benzyloxycarbonyl-3-methyl-4-methylene pyrrolidine-3-carboxylate(750 mg, 2.26 mmol) in dichloromethane (8 mL), trifluoroacetic acid (4mL) was added in an ice bath, and the mixture was stirred at roomtemperature for 4 hours. After concentrating the reaction mixture underreduced pressure, saturated aqueous solution of sodium hydrogencarbonate(20 mL) was added to the residue, and the mixture was washed withdiethylether (20 mL). To the aqueous layer was added 1N hydrochloricacid for acidification, and the solution was extracted with chloroform(100 mL×2). After drying the organic layer with anhydrous sodium sulfateand filtering, the filtrate was concentrated under reduced pressure toobtain 665 mg of the unpurified title compound as a colorless oilysubstance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.44 (3H, s), 3.23-3.36 (1H, m),4.11-4.30 (3H, m), 5.09-5.25 (4H, m), 7.27-7.39 (5H, m).

Reference Example 60(3R)-1-Benzyloxycarbonyl-3-(tert-butoxy-carbonylamino)-3-methyl-4-methylenePyrrolidine

To a solution of (3R)-1-benzyloxycarbonyl-3-methyl-4-methylenepyrrolidine-3-carboxylic acid (2.26 mmol) in toluene (20 mL),triethylamine (630 μl, 4.53 mmol) and diphenyl phosphoryl azide (536 μl,2.49 mmol) were added, and the mixture was stirred at room temperaturefor 1 hour. The mixture was heated under reflux in an oil bath at 110°C. for 1 hour. After concentrating the reaction mixture, the concentratewas dissolved in 1,4-dioxane (8 mL). 6N hydrochloric acid (4 mL) wasadded, and the mixture was stirred for 2 hours. Water (20 mL) was addedto the reaction mixture, and the mixture was washed with diethylether(50 mL). The aqueous layer was alkalized by adding saturated aqueoussolution of sodium hydrogencarbonate, and extracted with chloroform (100mL×2). After drying the organic layer with anhydrous sodium sulfate andfiltration, the filtrate was concentrated under reduced pressure, andthe residue was dissolved in toluene (8 mL). Di-tert-butyl dicarbonate(592 mg, 2.71 mmol) was in an ice bath, and the mixture was stirred atroom temperature for 67 hours. The reaction mixture was concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (hexane ethyl acetate, 5:1→3:1) to obtain 437 mg(62%) of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.42 (9H, s), 1.46 (3H, s), 3.56 (1H, t,J=9.2 Hz), 3.85-3.98 (1H, m), 4.13-4.26 (2H, m), 4.62-4.74 (1H, m),5.01-5.17 (4H, m), 7.27-7.38 (5H, m).

Reference Example 61(3R)-3-(tert-Butoxycarbonylamino)-3-methyl-4-methylene Pyrrolidine

Ammonia gas was bubbled into a solution of(3R)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-3-methyl-4-methylenepyrrolidine (469 mg, 1.41 mmol) in tetrahydrofuran (7 mL) at −78° C. toproduce a mixed solution of liquid ammonia and tetrahydrofuran (20 mL),and sodium (154 mg, 6.70 mmol) was added to this solution. The mixturewas stirred at the same temperature for 10 minutes. Saturated aqueoussolution of ammonium chloride (10 mL) was added at −78° C., and themixture was stirred at room temperature for 30 minutes. To the reactionmixture was added saturated aqueous solution of sodium hydrogencarbonate(30 mL), and the mixture was extracted with chloroform (100 mL×2). Theorganic layer was dried by anhydrous sodium sulfate, after filtration,the filtrate was concentrated under reduced pressure to obtain 255 mg(85%) of the title compound as colorless crystals.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.41-1.46 (12H, m), 2.90 (1H, d, J=11.7Hz), 3.45 (1H, d, J=14.2 Hz), 3.50-3.57 (1H, m), 3.69 (1H, d, J=16.1Hz), 4.64 (1H, br s), 5.01 (1H, t, J=2.2 Hz), 5.04 (1H, t, J=2.2 Hz).

Example 17 7-[(3R)-3-Amino-3-methyl-4-methylenepyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

To a solution of (3R)-3-(tert-butoxycarbonylamino)-3-methyl-4-methylenepyrrolidine (255 mg, 1.20 mmol) in dimethyl sulfoxide (3 mL),triethylamine (201 μl, 1.44 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (433 mg, 1.20 mmol) were added, and themixture was stirred at 35° C. for 15 hours. After concentrating thereaction mixture, a mixed solution of ethanol and water (ethanol:water,9:1) (15 mL) and triethylamine (0.5 mL) were added to the residue, andthe mixture was heated under reflux in an oil bath at 90° C. for 2hours. The reaction mixture was concentrated under reduced pressure, and10% aqueous solution of citric acid (50 mL) and water (50 mL) were addedto the residue. The mixture was extracted with ethyl acetate (100 mL×2),and the organic layer was washed with water (50 mL×3) and saturatedaqueous solution of sodium chloride (50 mL). The organic layer was driedwith anhydrous sodium sulfate and filtered, and the filtrate wasconcentrated under reduced pressure. The concentrate was dissolved inconcentrated hydrochloric acid (5 mL) in an ice bath, and the solutionwas stirred at room temperature for 10 minutes. Water (50 mL) was addedto the reaction mixture, and the mixture was washed with chloroform (100mL×3). 10 mol/l aqueous solution of sodium hydroxide was added to theaqueous layer in an ice bath to pH 11.0, and concentrated hydrochloricacid was added to adjust the pH to 7.4. The solution was then extractedwith chloroform (100 mL×5). The organic layer was dried with anhydroussodium sulfate and filtered, and the filtrate was concentrated underreduced pressure. The concentrate was purified by recrystallization fromethanol, and the crystals were dried under reduced pressure to obtain210 mg (43%) of the title compound as pale yellow crystals.

mp: 236-239° C. (decomposition).

[α]_(D)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.39 (3H, s), 1.41-1.66 (2H, m) 3.55(1H, dd, J=9.6, 3.2 Hz), 3.61 (3H, s), 3.63 (1H, d, J=10.0 Hz),4.02-4.07 (1H, m), 4.33 (2H, dd, J=25.9, 15.1 Hz), 4.99 (1H, d, J=63.7Hz), 5.05 (2H, t, J=1.7 Hz), 5.16 (2H, t, J=2.2 Hz), 7.68 (1H, d, J=14.2Hz), 8.45 (1H, d, J=2.0 Hz)

Elementary analysis for C₂₀H₂₁F₂N₃O₄:

Calculated: C, 59.25; H, 5.22; F, 9.37; N, 10.37.

Found: C, 59.14; H, 5.20; F, 9.62; N, 10.50.

IR (ATR) ν: 2960, 2856, 1716, 1618, 1547, 1514, 1452, 1369, 1327, 1306,1269, 1230, 1190, 1111 cm⁻¹.

Reference Example 62 Ethyl 3-[N-benzyloxycarbonyl-N-(ethoxycarbonylmethyl)amino]propionate

To a suspension of glycine ethyl ester hydrochloride (41.9 g, 0.3 mol)in ethanol (300 mL) was added triethylamine (41.8 mL, 0.3 mol) and ethylacrylate (10.8 mL, 0.1 mol) in an ice bath, and the mixture was stirredat 80° C. for 1 hour. The reaction mixture was concentrated underreduced pressure, and water (400 mL) was added to the residue, and themixture was extracted with ethyl acetate (200 mL×3). The extract waswashed with water (200 mL×2) and saturated aqueous solution of sodiumchloride (200 mL) in this order, an the dried with anhydrous sodiumsulfate. After the filtration, the filtrate was concentrated underreduced pressure, and the residue was dissolved in acetone (150 mL). Tothis solution were added aqueous solution of sodium carbonate (11.5 g,108 mmol) (50 mL) and a solution of benzyloxycarbonyl chloride (18.4 g,108 mmol) in acetone (50 mL) in an ice bath, and the mixture was stirredat room temperature for 2 hours. The reaction mixture was concentratedunder reduced pressure, and water (400 mL) was added to the residue. Themixture was extracted with ethyl acetate (200 mL×3), and the extract waswashed with saturated aqueous solution of sodium chloride (200 mL), anddried with anhydrous sodium sulfate. After the filtration, the filtratewas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (hexane:ethyl acetate, 5:1) to obtain31.1 g (92%) of the title compound as a colorless oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.16-1.29 (6H, m), 2.58-2.70 (2H, m),3.59-3.66 (2H, m), 4.04-4.22 (6H, m), 5.10-5.18 (2H, m), 7.26-7.38 (5H,m).

MS (ESI) m/z: 338 (M+H)⁺.

Reference Example 63 Ethyl1-benzyloxycarbonyl-4-oxopyrrolidine-3-carboxylate

To a solution of ethyl 3-[N-benzyloxycarbonyl-N-(ethoxycarbonylmethyl)amino]ethylpropionate (26.8 g, 79.5 mmol) in ethanol (200 mL),sodium ethoxide (20% solution in ethanol, 40.6 mL, 119.3 mmol) wasadded, and the mixture was heated under reflux for 2 hours. Afterconcentrating the reaction mixture under reduced pressure, the residuewas dissolved in water (100 mL). Concentrated hydrochloric acid wasadded to this solution in an ice bath for acidification, and thesolution was extracted with chloroform (100 mL×3). The extract waswashed with saturated aqueous solution of sodium chloride (100 mL), anddried with anhydrous sodium sulfate. After the filtration, the filtratewas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (hexane:ethyl acetate, 2:1) to obtain16.7 g (72%) of the title compound as a pale brown oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.25-1.33 (3H, m), 3.87-4.37 (7H, m),5.16-5.22 (2H, m), 7.23-7.41 (5H, m).

MS (ESI) m/z: 314 (M+Na)⁺.

Reference Example 64 Ethyl1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate

To a solution of ethyl1-benzyloxycarbonyl-4-oxopyrrolidine-3-carboxylate (1.0 g, 3.4 mmol) inacetone (30 mL), potassium carbonate (0.95 g, 6.9 mmol) and methyliodide (1 mL) were added, and the mixture was stirred at 45° C. for 1hour. The reaction mixture was concentrated under reduced pressure, andwater (20 mL) was added to the concentrate. The mixture was extractedwith ethyl acetate (20 mL×3). The extract was washed with 10% aqueoussolution of sodium thiosulfate (20 mL) and saturated aqueous solution ofsodium chloride (20 mL) in this order, and dried with anhydrous sodiumsulfate. After the filtration, the filtrate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (hexane:ethyl acetate, 4:1) to obtain 1.0 g (95%) of thetitle compound as a pale yellow oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.21 (3E, t, J=7.1 Hz), 1.55 (3H, s),3.53 (1H, d, J=11.7 Hz), 3.88 (1H, d, J=19.3 Hz), 4.07-4.20 (3H, m),4.37 (1H, d, J=12.0 Hz), 5.19 (2H, s), 7.30-7.40 (5H, m).

MS (ESI) m/z: 306 (M+H)⁺, 328 (M+Na)⁺.

Reference Example 65 Ethyl1-benzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate

To a solution of ethyl1-benzyloxycarbonyl-3-methyl-4-oxopyrrolidine-3-carboxylate (1.0 g, 3.28mmol) in methanol (20 mL), sodium borohydride (0.19 g, 4.92 mmol) wasadded at −20° C., and the mixture was stirred at the same temperaturefor 20 minutes. To the reaction mixture were added saturated aqueoussolution of ammonium chloride (20 mL) and water (20 mL), and the mixturewas extracted by ethyl acetate (20 mL×3). The extract was washed withsaturated aqueous solution of sodium chloride (20 mL), and dried withanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (hexane:ethyl acetate, 3:1) to obtain0.57 g (57%) of the title compound as a colorless oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.23-1.34 (6H, m), 2.90-3.87 (4H, m),4.11-4.56 (3H, m), 5.14 (2H, d, J=4.2 Hz), 7.24-7.40 (5H, m).

MS (ESI) m/z: 308 (M+H)⁺.

Reference Example 66 Ethyl1-benzyloxycarbonyl-4-methoxy-3-methylpyrrolidine-3-carboxylate

To a solution of ethyl1-benzyloxycarbonyl-4-hydroxy-3-methylpyrrolidine-3-carboxylate (0.55 g,1.8 mmol) in N,N-dimethylformamide (10 mL), methyl iodide (0.22 mL, 3.6mmol) and sodium hydride (55% in oil, 117 mg, 2.7 mmol) were added in anice bath, and the mixture was stirred at room temperature for 1 hour.Ice water was added to the reaction mixture, and the mixture wasextracted with ethyl acetate (20 mL×3). The extract was washed withwater (20 mL×3) and saturated aqueous solution of sodium chloride (20mL) in this order, and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe residue was purified by silica gel column chromatography(hexane:ethyl acetate, 2:1) to obtain 0.38 g (66%) of the title compoundas a pale yellow oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.24-1.29 (6H, m), 3.19-3.73 (3H, m),3.30 (3H, s), 3.80-4.28 (4H, m), 5.14 (2H, s), 7.29-7.39 (5H, m).

MS (ESI) m/z: 322 (M+H)⁺.

Reference Example 671-Benzyloxycarbonyl-4-methoxy-3-methylpyrrolidine-3-carboxylic Acid

To a solution of ethyl1-benzyloxycarbonyl-4-methoxy-3-methylpyrrolidine-3-carboxylate (0.38 g,1.18 mmol) in ethanol (4 mL) was added 1N aqueous solution of sodiumhydroxide (4 mL), and the mixture was stirred at 40° C. for 17.5 hours.Water (10 mL) was added to the reaction mixture, and the mixture waswashed with ethyl acetate (10 mL×2). To the aqueous layer was added 1Nhydrochloric acid (10 mL), and the mixture was extracted by chloroform(20 mL×3). The extract was washed with saturated aqueous solution ofsodium chloride (20 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure toobtain 0.31 g (89%) of the title compound as a colorless oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.33 (3H, s), 3.34-3.47 (1H, m), 3.36(3H, s), 3.53-3.75 (2H, m), 3.85-3.95 (1H, m), 4.09-4.18 (1H, m), 5.14(1H, s), 7.25-7.40 (5H, m).

MS (ESI) m/z: 294 (M+H)⁺, 316 (M+Na)⁺.

Reference Example 681-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine

To a solution of1-benzyloxycarbonyl-4-methoxy-3-methylpyrrolidine-3-carboxylic acid (0.3g, 1.02 mmol) in toluene (10 mL), triethylamine (0.29 mL, 2.05 mmol) anddiphenyl phosphoryl azide (0.24 mL, 1.13 mmol) were added, and themixture was stirred at 125° C. for 2 hours. After concentrating thereaction mixture under reduced pressure, 1,4-dioxane (4 mL), water (4mL) and concentrated hydrochloric acid (1 mL) were added to the residue,and the mixture was stirred at 50° C. for 2 hours. Water (10 mL) wasadded to the reaction mixture, and the mixture was washed with ethylacetate (10 mL). The aqueous layer was alkalized with 10N sodiumhydroxide aqueous solution, and extracted by chloroform (20 mL×3). Theextract was washed with saturated aqueous solution of sodium chloride(20 mL), and dried with anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure, and the residue wasdissolved in ethanol (10 mL). Di-tert-butyl dicarbonate (0.27 g, 1.22mmol) was added to the solution, and the mixture was stirred at 50° C.for 1 hour. The reaction mixture was concentrated under reducedpressure, and the residue was purified by silica gel columnchromatography (hexane:ethyl acetate, 6:1) to obtain 0.27 g (72%) ofracemic body of the title compound as a colorless oily product.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.41-1.45 (12H, m), 3.30-3.82 (7H, m),4.12 (1H, q, J=7.1 Hz), 5.09-5.26 (3H, m), 7.24-7.38 (5H, m).

MS (ESI) m/z: 387 (M+Na)⁺.

Next, the racemic body of the title compound (0.68 g, 1.87 mmol) wassubjected to high-performance liquid chromatography using an opticallyactive column, and fraction α (0.29 g, 43%) and fraction β (0.28 g, 41%)which are enantiomers of the title compound were respectively obtainedas colorless oily products.

Conditions of the Optical Resolution:

Column: CHIRALPAK AD (DAICEL, 20 mm×250 mm)

solvent: 2-propanol:hexane, 1:9

Flow rate: 10 ml/min

Detection: UV (254 nm)

Retention time: about 18.1 minutes (fraction α), about 23.5 minutes(fraction β)

Reference Example 693-(tert-Butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine (Derived fromFraction α)

To a solution of1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine(fraction α) (0.29 g, 0.8 mmol) in methanol (10 mL), 5% palladium-carboncatalyst (water content, 50%; 0.15 g) was added, and the suspension wasstirred at room temperature for 16.5 hours in hydrogen atmosphere. Thereaction mixture was filtered through celite, and the filtrate wasconcentrated under reduced pressure to obtain the title compound as acolorless oily product. This product was used in the subsequent reactionwith no further purification.

MS (ESI) m/z: 231 (M+H)⁺.

Example 187-(3-Amino-4-methoxy-3-methylpyrrolidine-1-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid (Substituent at Position 7 Derived from Fraction α)

To a solution of3-(tert-butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine in dimethylsulfoxide (2 mL), triethylamine (0.33 mL, 2.4 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (0.19 g, 0.53 mmol) were added, and themixture was stirred at 40° C. for 21.5 hours in nitrogen atmosphere. Tothe reaction mixture were added ethanol containing 10% water (10 mL) andtriethylamine (1 mL), and the mixture was heated under reflux for 1hour. The reaction mixture was concentrated under reduced pressure, andethyl acetate (20 mL) and 10% aqueous solution of citric acid (20 mL)were added to the concentrate for separation into two layers. Theaqueous layer was extracted with ethyl acetate (20 mL×2), and theorganic layers were combined and washed with water (20 mL×3) andsaturated aqueous solution of sodium chloride (20 mL) in this order anddried with anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (chloroform methanol, 50:1). The eluatewas concentrated under reduced pressure, and to the concentrate wasadded concentrated hydrochloric acid (1 mL) in an ice bath. The reactionmixture was stirred at room temperature for 15 minutes, and washed withchloroform (10 mL×5). 10N aqueous solution of sodium hydroxide was addedto the aqueous layer to pH 12.0, and hydrochloric acid was then added toadjust the pH to 7.4. The mixture was extracted with chloroform (30mL×5) and dried with anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure. The concentrate waspurified by recrystallization from ethanol-diethylether, and thecrystals were dried under reduced pressure to obtain 103 mg (46%) of thetitle compound as a colorless crystalline powder.

mp: 150-151° C.

¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 1.21 (3H, s), 1.47-1.58 (2H, m)3.24-3.64 (4H, m), 3.31 (3H, s), 3.37 (3H, s), 3.87-3.94 (1H, m) 4.08(1H, dd, J=12.3, 7.2 Hz), 4.96-5.18 (1H, m), 7.67 (1H, d, J=13.9 Hz),8.63 (1H, d, J=2.7 Hz).

Elementary analysis for C₂₀H₂₃F₂N₃O₅:

Calculated: C, 56.73; H, 5.48; F, 8.97; N, 9.92.

Found: C, 56.71; H, 5.54; F, 9.03; N, 9.73.

IR (ATR) ν: 2931, 1718, 1617, 1513, 1450, 1438 cm⁻¹.

MS (FAB) m/z: 424 (M+H)⁺.

Reference Example 703-(tert-Butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine (Derived fromFraction β)

To a solution of1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine(fraction β) (0.28 g, 0.77 mmol) in methanol (10 mL) was added 5%palladium-carbon (containing water, 0.14 g), and the mixture was stirredat room temperature for 18 hours in hydrogen atmosphere. The reactionmixture was filtered through celite, and the filtrate was concentratedunder reduced pressure to obtain the title compound as a colorless oilyproduct. This product was used in the subsequent reaction withoutfurther purification.

(ESI) m/z: 231 (M+H)⁺.

Example 197-(3-Amino-4-methoxy-3-methylpyrrolidine-1-yl)-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid (Substituent at Position 7 Derived from Fraction β)

To a solution of3-(tert-butoxycarbonylamino)-4-methoxy-3-methylpyrrolidine in dimethylsulfoxide (1 mL), triethylamine (0.33 mL, 2.4 mmol) and6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (0.19 g, 0.53 mmol) were added, and themixture was stirred at 40° C. for 3 days in nitrogen atmosphere. To thereaction mixture were ethanol containing 10% water (10 mL) andtriethylamine (1 mL), and the mixture was heated under reflux for 1hour. After concentrating the reaction mixture under reduced pressure,ethyl acetate (20 mL) and 10% aqueous solution of citric acid (20 mL)were added to the concentrate for separation into two layers. Theaqueous layer was extracted with ethyl acetate (20 mL×2), and theorganic layers were combined and washed with water (20 mL×3) andsaturated aqueous solution of sodium chloride (20 mL) in this order, anddried with anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (chloroform:methanol, 100:1). Theeluate was concentrated under reduced pressure, and concentratedhydrochloric acid (2 mL) was added to the residue in an ice bath. Themixture was stirred at room temperature for 20 minutes, and the reactionmixture was washed with chloroform (10 mL×5). 10N aqueous solution ofsodium hydroxide was added to the aqueous layer to pH 12.0, andhydrochloric acid was then added to adjust the pH to 7.4. The solutionwas extracted with chloroform (30 mL×5) and dried with anhydrous sodiumsulfate. After filtration, the filtrate was concentrated under reducedpressure, and the residue was purified by recrystallization fromethanol-diethylether-hexane. The crystals were dried under reducedpressure to obtain 56 mg (25%) of the title compound as a pale yellowcrystalline powder.

mp: 162-163° C.

¹H-NMR (400 MHz, DMSO-d₆) δ ppm: 1.20 (3H, s), 1.54-1.66 (2H, m),3.19-3.59 (4H, m), 3.30 (3H, s), 3.56 (3H, s), 3.87-3.96 (1H, m),4.05-4.14 (1H, m), 4.82-5.06 (1H, m), 7.66 (1H, d, J=13.9 Hz), 8.68 (1H,s).

Elementary analysis for C₂₀H₂₃F₂N₃O₅:

Calculated: C, 56.73; H, 5.48; F, 8.97; N, 9.92.

Found: C, 56.56; H, 5.49; F, 9.09; N, 9.80.

IR (ATR) ν: 2937, 1725, 1621, 1511, 1436 cm⁻¹.

MS (FAB) m/z: 424 (M+H)⁺.

Reference Example 71 Ethyl(3R,4R)-4-fluoromethyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of(4R)-4-fluoromethyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine (34.1 g, 154mmol) and ethyl chloroformate (16.1 mL, 169 mmol) in tetrahydrofuran(500 mL), lithium bistrimethylsilyl amide (323 mL, 323 mmol, 1.0Msolution in tetrahydrofuran) was added at 0° C., and the mixture wasstirred at the same temperature for 30 minutes. Saturated aqueoussolution of ammonium chloride (700 mL) was added to reaction mixture atthe same temperature, and the mixture was extracted with ethyl acetate(700 mL×2). The organic layer was washed with saturated aqueous solutionof sodium chloride (500 mL), and dried with anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure,and the residue was purified by silica gel column chromatography(elution with hexane:ethyl acetate, 5:1→2:3) to obtain 35.0 g (77%) ofthe title compound as a pale yellow oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.31 (3H, t, J=7.1 Hz), 1.55 (3H, d,J=7.1 Hz), 2.88-3.01 (1H, m), 3.17 (1H, d, J=7.4 Hz), 3.39 (1H, d, J=7.4Hz), 3.74 (1H, t, J=6.1 Hz), 4.26 (2H, q, J=7.1 Hz), 4.42 (2H, dd,J=47.1, 5.1 Hz), 5.49 (1H, q, J=7.1 Hz), 7.26-7.38 (5H, m)

Reference Example 72 Ethyl(3S,4R)-4-fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of ethyl(3R,4R)-4-fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(33.4 g, 114 mmol) and iodomethane (9.94 mL, 159 mmol) intetrahydrofuran (670 mL), potassium bistrimethylsilyl amide (274 mL, 137mmol, 0.5M solution in toluene) was added at −78° C., and the mixturewas stirred at the same temperature for 10 minutes. The temperature wasgradually increased in 30 minutes with stirring to −10° C., andsaturated aqueous solution of ammonium chloride (700 mL) was added tothe reaction mixture. The mixture was extracted with ethyl acetate (700mL×2), and the organic layer was washed with saturated aqueous solutionof sodium chloride (500 mL), and dried with anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure,and the residue was purified by silica gel column chromatography(elusion by hexane:ethyl acetate, 7:1→1:1) to obtain 29.1 g (83%) of thetitle compound as a pale yellow oily substance.

—H-NMR (400 MHz, CDCl₃) δ ppm: 1.29 (3H, t, J=7.1 Hz), 1.49 (3H, s),1.56 (3H, t, J=8.8 Hz), 2.34-2.47 (1H, m), 3.04 (1H, dd, J=9.3, 8.1 Hz),3.29 (1H, t, J=9.5 Hz), 4.19 (2H, q, J=7.1 Hz), 4.34-4.59 (2H, m), 5.52(1H, q, J=7.0 Hz), 7.28-7.39 (5H, m)

Reference Example 73(3S,4R)-4-Fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylicAcid

To a solution of ethyl(3S,4R)-4-fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(20.0 g, 65.1 mmol) in ethanol (400 mL), 10 mol/l aqueous solution ofsodium hydroxide (65.1 mL, 651 mmol) was added dropwise, and the mixturewas stirred for 30 minutes. Water (400 mL) was added to the reactionmixture in an ice bath, and the aqueous solution was washed withdiethylether (500 mL). Concentrated hydrochloric acid was added to theaqueous layer in an ice bath to pH 2 to 3, and the mixture was extractedwith chloroform (500 mL×3). The organic layer was dried with anhydroussodium sulfate, and after filtration, the filtrate was concentratedunder reduced pressure. The concentrate was azeotropically distilled byadding toluene (20 mL), and dried under reduced pressure to obtain 19.16g (quantitative) of the title compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.51 (3H, s), 1.59 (3H, d, J=7.1 Hz),2.42-2.54 (1H, m), 3.12 (1H, dd, J=10.5, 7.1 Hz), 3.33 (1H, dd, J=10.5,3.9 Hz), 4.60 (2H, dd, J=46.8, 5.1 Hz), 5.49 (1H, q, J=7.0 Hz),7.26-7.40 (5H, m).

Reference Example 74(3S,4S)-3-(tert-Butoxycarbonylamino)-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine

To a solution of(3S,4R)-4-fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylicacid (65.1 mmol) and diphenylphosphoryl azide (15.4 mL, 71.6 mmol) intoluene (380 mL), triethylamine (18.2 mL, 130 mmol) was added, and themixture was stirred in an oil bath at 110° C. for 1 hour. The reactionmixture was concentrated under reduced pressure to obtain the crudeproduct in the form of an isocyanate.

MS (ESI) m/z: 277 (M+H)+

The resulting crude product in the form of an isocyanate was dissolvedin 1,4-dioxane (90 mL), and water (45 mL) and concentrated hydrochloricacid (45 mL) was added in an ice bath, and the mixture was stirred atroom temperature for 3 hours. Water (180 mL) was added to the reactionmixture, and the mixture was washed with diethylether (200 mL). 10 mol/laqueous solution of sodium hydroxide was added to the aqueous layer topH 9 to 10 in an ice bath, and the mixture was extracted with chloroform(500 mL×2). The organic layer was washed with saturated aqueous solutionof sodium chloride (100 mL), and after drying with anhydrous sodiumsulfate and filtration, the filtrate was concentrated under reducedpressure to obtain the crude product in the form of an amine (10.1 g,DM041701).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.30 (3H, s), 1.55 (3H, d, J=7.1 Hz),2.18-2.32 (1H, m), 2.98 (1H, dd, J=10.3, 7.3 Hz), 3.17 (1H, dd, J=10.4,4.3 Hz), 4.48-4.72 (2H, m), 5.48 (1H, q, J=7.1 Hz), 7.15-7.37 (5H, m).

MS (ESI) m/z: 251 (M+H)⁺.

The crude product in the form of an amine (10.1 g, 40.2 mmol) wasdissolved in toluene (200 mL), and a solution of 65% (by weight)solution of sodium bis(2-methoxyethoxy)aluminum hydride in toluene (48.3mL, 161 mmol) in toluene (6 mL) was added dropwise in 15 minutes in anice bath so that the inner temperature does not exceed 50° C., and themixture was stirred at room temperature for 10 minutes. The reactionmixture was cooled in an ice bath, and 25% (by weight) aqueous solutionof sodium hydroxide (160 mL) was added dropwise. After quenching thesolution, the solution was extracted with toluene (135 mL). The organiclayer was washed with saturated aqueous solution of sodium chloride (100mL), and the organic layer was dried with anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressureto obtain the crude product in the form of an amine (10.0 g).

MS (ESI) m/z: 237 (M+H)+

To the crude product in the form of an amine (10.0 g, 40.2 mmol) wasadded di-tert-butyl dicarbonate (9.65 g, 44.2 mmol), and the reactionmixture was stirred at room temperature for 10 hours. After stirring,the solvent was removed by distillation under reduced pressure, and theresidue was purified by silica gel column chromatography (eluted withhexane:ethyl acetate; 19:1→5:4) to obtain 1.78 g (5 steps, 8%) of thetitle compound as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.32 (3H, d, J=6.6 Hz), 1.42 (9H, s),1.52 (3H, s), 2.35 (1H, td, J=14.2, 7.1 Hz), 2.45 (1H, t, J=8.3 Hz),2.57 (1H, d, J=9.3 Hz), 2.70 (1H, d, J=9.3 Hz), 2.92 (1H, t, J=8.8 Hz),3.31 (1H, q, J=6.6 Hz), 4.42 (1H, ddd, J=47.1, 9.3, 6.1 Hz), 4.63 (1H,ddd, J=47.4, 9.3, 6.1 Hz), 4.94 (1H, s), 7.19-7.31 (5H, m).

MS (ESI) m/z: 337 (M+H)⁺.

Reference Example 75(3S,4S)-3-(tert-Butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine

To a solution of(3S,4S)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine(1.35 g, 4.01 mmol) in ethanol (30 mL) was added 10% palladium-carboncatalyst (containing 52.8% water, 1.30 g), and the suspension wasstirred in an oil bath at 40° C. for 12 hours in hydrogen gasatmosphere. After removing the catalyst by filtration, the filtrate wasconcentrated under reduced pressure to obtain 932 mg (quantitative) ofthe crude target compound as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.44 (9H, s), 1.47 (3H, s), 2.16-2.30(1H, m), 2.85 (1H, d, J=11.5 Hz), 3.01 (1H, dd, J=11.3, 7.4 Hz), 3.20(1H, dd, J=11.3, 8.6 Hz), 3.29 (1H, d, J=11.8 Hz), 4.49-4.69 (2H, m),4.98 (1H, s).

MS (ESI) m/z: 233 (M+H)⁺.

Example 207-[(3S,4S)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S,4S)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(221 mg, 0.951 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (343 mg, 0.951 mmol), and triethylamine(0.159 mL, 1.14 mmol) were dissolved in dimethyl sulfoxide (3 mL), andstirred in an oil bath at 35° C. for 18 hours. After concentrating thereaction mixture, a mixed solution of ethanol and water (ethanol:water,9:1) (40 mL) and triethylamine (1 mL) were added to the concentrate, andthe mixture was heated under reflux for 2 hours. The reaction mixturewas concentrated under reduced pressure. The concentrate was dissolvedin ethyl acetate (100 mL×2), and the solution was washed with 10%aqueous solution of citric acid (50 mL), water (50 m×3), and saturatedaqueous solution of sodium chloride (100 mL). The organic layer wasdried with anhydrous sodium sulfate, and after filtering the residue,the filtrate was concentrated under reduced pressure. The concentratewas dissolved in concentrated hydrochloric acid (20 mL), and thesolution was stirred at room temperature for 30 minutes. The reactionmixture was washed with chloroform (100 mL×5), and to the aqueous layerwas added 10 mol/l aqueous solution of sodium hydroxide to pH 12 in anice bath. Hydrochloric acid was added to adjust the pH to 7.4, and thesolution was extracted with chloroform (150 mL×4). The organic layer wasdried with anhydrous sodium sulfate, and after filtration, the filtratewas concentrated under reduced pressure. The concentrate was purified byrecrystallization from ethanol, and the crystals were dried underreduced pressure to obtain 269 mg (24%) of the title compound as a paleyellow powder.

mp: 187-189° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.33 (3H, s), 1.40-1.51 (1H, m),1.51-1.63 (1H, m), 2.45-2.58 (1H, m), 3.49 (1H, d, J=9.6 Hz), 3.58 (3H,s), 3.64-3.73 (2H, m), 3.85 (1H, t, J=9.4 Hz), 4.00-4.05 (1H, m), 4.69(1H, ddd, J=37.3, 10.0, 5.9 Hz), 4.80-4.84 (1H, m), 5.00 (1H, d, J=64.0Hz), 7.66 (1H, d, J=14.5 Hz), 8.42 (1H, d, J=2.7 Hz).

Elementary analysis for C₂₀H₂₂F₃N₃O₄0.25H₂O:

Calculated: C, 55.88; H, 5.28; F, 13.26; N, 9.77.

Found: C, 55.58; H, 5.29; F, 13.17; N, 9.84.

IR (ATR) ν: 2962, 2873, 1720, 1618, 1510, 1435, 1363, 1311, 1275, 1234,1186, 1122 cm⁻¹.

Example 217-[(3S,4S)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S,4S)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(46.5 mg, 0.200 mmol),1-cyclopropyl-6,7-difluoro-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (68.6 mg, 0.200 mmol), and triethylamine(0.0335 mL, 0.240 mmol) were dissolved in dimethyl sulfoxide (0.5 mL),and the mixture was stirred in an oil bath at 35° C. for 19 hours. Afterconcentrating the mixture, a mixed solution of ethanol and water(ethanol:water, 9:1) (20 mL) and triethylamine (0.5 mL) added to theconcentrate, and the mixture was heated under reflux for 2 hours. Thereaction mixture was concentrated under reduced pressure, and theresidue was dissolved in ethyl acetate (50 mL×2), and the solution waswashed with 10% aqueous solution of citric acid (50 mL), water (50 m×3),and saturated aqueous solution of sodium chloride (50 mL). The organiclayer was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The residue wasdissolved in concentrated hydrochloric acid (5 μL) in an ice bath, andthe solution was stirred at room temperature for 30 minutes. Thereaction mixture was washed with chloroform (100 mL×5), and to theaqueous layer was added 10 mol/l aqueous solution of sodium hydroxide topH 12 in an ice bath, and the hydrochloric acid was added to adjust thepH to 7.4. The solution was extracted with chloroform (100 mL×3), andthe organic layer was dried with anhydrous sodium sulfate, and afterfiltration, the filtrate was concentrated under reduced pressure. Theconcentrate was purified by recrystallization from ethanol, and thecrystals were dried under reduced pressure to obtain 42.6 mg (52%) ofthe title compound as a white powder.

mp: 226-229° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.32-0.90 (1H, m), 0.98-1.11 (2H, m),1.14-1.22 (1H, m), 1.39 (3H, s), 2.47-2.60 (1H, m), 3.44 (1H, d, J=10.0Hz), 3.58 (3H, s), 3.73 (1H, dd, J=10.4, 2.6 Hz), 3.78 (2H, d, J=8.3Hz), 4.04-4.11 (1H, m), 4.79-4.88 (2H, m), 7.65 (1H, d, J=14.4 Hz), 8.49(1H, s).

Elementary analysis for C₂₀H₂₃F₂N₃O₄:

Calculated: C, 58.96; H, 5.69; F, 9.33; N, 10.31.

Found: C, 58.90; H, 5.70; F, 9.33; M, 10.19.

IR (ATR) ν: 3450, 3374, 3079, 2962, 2873, 1724, 1620, 1508, 1439, 1373,1315, 1273, 1228, 1186, 1153, 1109 cm¹.

Example 227-[(3S,4S)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S,4S)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(932 mg, 4.01 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (922 mg, 2.67 mmol), and triethylamine (0.447mL, 3.20 mmol) was dissolved in sulfolane (5 mL), and the mixture wasstirred in an oil bath at 35° C. for 166 hours. After concentrating thereaction mixture, a mixed solution of ethanol and water (ethanol:water,9:1) (80 mL) and triethylamine (1 mL) were added to the concentrate, andthe mixture was stirred in an oil bath at 90° C. for 30 minutes. Thereaction mixture was concentrated under reduced pressure, and theconcentrate was dissolved in ethyl acetate (200 mL×2), and washed with10% aqueous solution of citric acid (100 mL), water (100 m×3), andsaturated aqueous solution of sodium chloride (100 mL). The organiclayer was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The residue was purifiedby short silica gel column chromatography (elusion bychloroform:methanol, 49:1→9:1). The residue was dissolved inconcentrated hydrochloric acid (20 mL) in an ice bath, and the mixturewas stirred at room temperature for 30 minutes, and washed withchloroform (100 mL×5). To the aqueous layer was added 10 mol/l aqueoussolution of sodium hydroxide to pH 12 in an ice bath, and hydrochloricacid was added to adjust the pH to 7.4. The solution was extracted withchloroform (150 mL×4), and the organic layer was dried with anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure, and the residue was purified by recrystallization fromethanol. The crystals were dried under reduced pressure to obtain 97.7mg (9%) of the title compound as a pale yellow powder.

mp: 135-137° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.18-1.32 (1H, m), 1.38 (3H, s),1.55-1.66 (1H, m), 2.52 (3H, s), 2.53-2.65 (1H, m), 3.20 (1H, d, J=9.8Hz), 3.49 (1H, t, J=9.2 Hz), 3.70 (1H, t, J=8.9 Hz), 3.78 (1H, dd,J=9.8, 3.2 Hz), 4.09 (1H, dt, J=9.9, 4.4 Hz), 4.70 (1H, ddd, J=41.9,9.4, 6.0 Hz), 4.80-4.86 (1H, m), 5.01 (1H, d, J=67.9 Hz), 7.69 (1H, d,J=14.0 Hz), 8.45 (1H, d, J=3.2 Hz).

Elementary analysis for C₂₀H₂₂F₃N₃O₄.0.7H₂O:

Calculated: C, 56.80; H, 5.60; N, 9.94.

Found: C, 56.52; H, 5.53; N, 10.06.

IR (ATR) ν: 2968, 2873, 1718, 1614, 1508, 1462, 1431, 1396, 1358, 1319,1282, 1163, 1128, 1101 cm⁻¹.

Example 237-[(3S,4S)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S,4S)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(298 mg, 1.28 mmol),1-cyclopropyl-6,7-difluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (280 mg, 0.855 mmol), and triethylamine(0.143 mL, 1.03 mmol) were dissolved in sulfolane (1.5 mL), and themixture was stirred in an oil bath at 35° C. for 142 hours. The reactionmixture was concentrated, and a mixed solution of ethanol and water(ethanol:water, 9:1) (40 μL) and triethylamine (1 mL) were added to theconcentrate, and the mixture was stirred in an oil bath at 90° C. for 30minutes. The reaction mixture was concentrated under reduced pressure,and the residue was dissolved in ethyl acetate (100 mL×2), and washedwith 10% aqueous solution of citric acid (50 mL), water (50 m×3) andsaturated aqueous solution of sodium chloride (50 mL). The organic layerwas dried with anhydrous sodium sulfate, and after filtration, thefiltrate was concentrated under reduced pressure. The concentrate waspurified by short silica gel column chromatography (elusion bychloroform-methanol, 49:1→9:1), and the residue was dissolved inconcentrated hydrochloric acid (10 mL). After stirring the solution atroom temperature for 30 minutes, the reaction mixture was washed withchloroform (50 mL×5). To the aqueous layer was added 10 mol/l aqueoussolution of sodium hydroxide to pH 12 in an ice bath, and hydrochloricacid was added to adjust the pH to 7.4. The solution was extracted withchloroform (100 mL×4), and the organic layer was dried with anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure. The concentrate was purified by recrystallization fromethanol, and the crystals were dried under reduced pressure to obtain109 mg (33%) of the title compound as a pale yellow powder.

mp: 212-215° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.75-0.90 (2H, m), 1.10-1.25 (2H, m),1.38 (3H, s), 2.56-2.61 (1H, m), 2.58 (3H, s), 3.29 (1H, d, J=9.6 Hz),3.53-3.63 (2H, m), 3.64 (1H, dd, J=9.8, 2.7 Hz), 4.10-4.17 (1H, m), 4.68(1H, ddd, J=34.7, 10.2, 5.3 Hz), 4.80-4.82 (1H, m), 7.67 (1H, d, J=14.0Hz), 8.57 (1H, s).

Elementary analysis for C₂₀H₂₃F₂N₃O₃:

Calculated: C, 61.37; H, 5.92; F, 9.71; N, 10.74.

Found: C, 61.26; H, 5.91; F, 9.86; N, 10.72.

IR (ATR) ν: 3361, 3087, 2974, 2873, 171:2, 1616, 1545, 1508, 1458, 1431,1358, 1315, 1228, 1188, 1151, 1109 cm⁻¹.

Reference Example 76(3S,4S)-3-Ethoxycarbonyl-4-fluoromethyl-1-[(1R)-1-phenylethyl]-2-pyrrolidone

To a solution of(4S)-4-fluoromethyl-1-[(1R)-1-phenylethyl]-2-pyrrolidone (7.59 g, 34.3mmol) and ethyl chloroformate (3.92 mL, 41.2 mmol) in tetrahydrofuran(150 mL), lithium bistrimethylsilyl amide (75.5 mL, 75.5 mmol, 1.0Msolution in tetrahydrofuran) was added at 0° C., and the mixture wasstirred the same temperature for 20 minutes. Saturated aqueous solutionof ammonium chloride (200 mL) was added to the reaction mixture at thesame temperature, and the mixture was extracted with ethyl acetate (200mL×2). The organic layer was washed with saturated aqueous solution ofsodium chloride (100 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe residue was purified by silica gel column chromatography (elusion byhexane:ethyl acetate, 5:1→2:3) to obtain 8.50 g (85%) of the titlecompound as a pale yellow oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.33 (3H, t, J=7.2 Hz), 1.56 (3H, d,J=7.4 Hz), 2.80 (1H, dd, J=9.8, 6.1 Hz), 2.94-3.11 (1H, m), 3.34 (1H, d,J=7.1 Hz), 3.53 (1H, t, J=9.1 Hz), 4.23-4.31 (3H, m), 4.37 (1H, dd,J=5.6, 2.7 Hz), 5.49 (1H, q, J=7.1 Hz), 7.26-7.37 (5H, m).

Reference Example 77(3R,4S)-3-Ethoxycarbonyl-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]-2-pyrrolidone

To a solution of(3S,4S)-3-ethoxycarbonyl-4-fluoromethyl-1-[(1R)-1-phenylethyl]-2-pyrrolidone(8.30 g, 28.3 mmol) and iodomethane (2.47 mL, 39.6 mmol) intetrahydrofuran (170 mL), potassium bistrimethylsilyl amide (67.9 mL,34.0 mmol, 0.5M solution in toluene) was added at −78° C., and themixture was stirred at the same temperature for 10 minutes. Whilestirring, the temperature was gradually increased to −10° C. in 30minutes. Saturated aqueous solution of ammonium chloride (200 mL) wasadded to the reaction mixture, and the mixture was extracted with ethylacetate (200 mL×2), and the organic layer was washed with saturatedaqueous solution of sodium chloride (150 mL) and dried with anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (elusion by hexane:ethyl acetate, 7:1→1:1) to obtain 7.91g (91%) of the title compound as a pale yellow oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.10 (3H, t, J=7.2 Hz), 1.50 (3H, s),1.57 (3H, dd, J=7.1, 2.4 Hz), 2.46-2.60 (1H, m), 2.89 (1H, t, J=9.5 Hz),3.35 (1H, dd, J=9.4, 7.9 Hz), 4.00-4.15 (2H, m), 4.20-4.45 (2H, m), 5.56(1H, q, J=7.3 Hz), 7.26-7.37 (5H, m)

Reference Example 78(3R,4S)-4-Fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylicAcid

To a solution of(3R,4S)-3-ethoxycarbonyl-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]-2-pyrrolidone(1.05 g, 3.42 mmol) in ethanol (20 mL), 10 mol/l aqueous solution ofsodium hydroxide (3.42 mL, 34.2 mmol) was added dropwise in an ice bath,and the mixture was stirred for 30 minutes. Water (20 mL) was added tothe reaction mixture in an ice bath, and the aqueous solution was washedwith diethylether (50 mL). To the aqueous layer was added concentratedhydrochloric acid to pH 2 to 3 in an ice bath, and the solution wasextracted with chloroform (50 mL×3). The organic layer was washed withsaturated aqueous solution of sodium chloride (50 mL), and dried withanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled by adding toluene (20 mL), and dried under reduced pressure toobtain 950 mg (99%) of the title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.53-1.61 (6H, m), 2.50-2.61 (1H, m),3.02 (1H, dd, J=10.5, 3.4 Hz), 3.49 (1H, dd, J=10.5, 7.1 Hz), 4.34-4.56(2H, m), 5.49 (1H, q, J=7.0 Hz), 7.14-7.36 (5H, m).

MS ESI) m/z: 280 (M+H)⁺.

Reference Example 79(3R,4R)-3-(tert-Butoxycarbonylamino)-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine

To a solution of(3R,4S)-4-fluoromethyl-3-methyl-2-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylicacid (950 mg, 3.40 mmol) and diphenylphosphoryl azide (0.806 mL, 3.74mmol) in toluene (20 mL), triethylamine (0.948 mL, 6.80 mmol) was added,and the mixture was stirred in an oil bath at 110° C. for 1 hour. Thereaction mixture was concentrated under reduced pressure to obtain thecrude product in the form of an isocyanate.

MS (ESI) m/z: 277 (M+H)+

The crude product in the form of an isocyanate was dissolved in1,4-dioxane (5 mL), and water (2.5 mL) and concentrated hydrochloricacid (2.5 mL) were added to this solution in an ice bath. After stirringthe mixture at room temperature for 13 hours, water (10 mL) was added tothe reaction mixture, and the mixture was washed with diethylether (50mL). To the aqueous layer was added 10 mol/l aqueous solution of sodiumhydroxide to pH 9 to 10 in an ice bath, and the solution was extractedwith chloroform (100 mL×3). The organic layer was washed with saturatedaqueous solution of sodium chloride (100 mL), and dried with anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure to obtain the crude product in the form of an amine(470 mg)

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.36 (3H, s), 1.54 (3H, d, J=7.1 Hz),2.29-2.42 (1H, m), 2.82 (1H, dd, J=10.3, 4.4 Hz), 3.35 (1H, dd, J=10.1,7.4 Hz), 4.32-4.62 (2H, m), 5.47 (1H, q, J=7.0 Hz), 7.26-7.37 (5H, m).

MS (ESI) m/z: 251 (M+H)⁺.

The crude product in the form of an amine (470 mg, 1.88 mmol) wasdissolved in toluene (10 mL), and a solution of 65% (by weight) solutionof sodium bis(2-methoxyethoxy)aluminum hydride in toluene (2.25 mL, 7.52mmol) in toluene (2 mL) was added dropwise in 15 minutes in an ice bathso that the inner temperature does not exceed 50° C., and the mixturewas stirred at room temperature for 10 minutes. The reaction mixture wascooled in an ice bath, and 25% (by weight) aqueous solution of sodiumhydroxide (5 mL) was added dropwise. After quenching the solution, thesolution was extracted with toluene (40 mL). The organic layer was driedwith anhydrous sodium sulfate, and the organic layer was dried withanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to obtain the crude product in theform of an amine (490 mg).

MS (ESI) m/z: 237 (M+H)+

To the crude product in the form of an amine (490 mg, 1.88 mmol) wasadded di-tert-butyl dicarbonate (451 mg, 2.07 mmol), and the reactionmixture was stirred at room temperature for 17 hours. Afterconcentrating the reaction mixture under reduced pressure, the residuewas purified by silica gel column chromatography (elusion byhexane:ethyl acetate, 19:1→5:4) to obtain 404 mg (5 steps, 35%) of thetitle compound as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (3H, d, J=6.6 Hz), 1.44 (9H, s),1.53 (3H, s), 2.31 (1H, td, J=14.3, 7.2 Hz), 2.52 (1H, t, J=8.4 Hz),2.59 (1H, d, J=9.0 Hz), 2.69-2.77 (2H, m), 3.28 (1H, q, J=6.6 Hz), 4.42(1H, ddd, J=47.2, 9.4, 6.1 Hz), 4.62 (1H, ddd, J=47.4, 9.4, 6.2 Hz),4.98 (1H, s), 7.22-7.30 (5H, m).

MS (ESI) m/z: 337 (M+H)⁺.

Reference Example 80(3R,4R)-3-(tert-Butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine

To a solution of(3R,4R)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methyl-1-[(1R)-1-phenylethyl]pyrrolidine(250 mg, 0.743 mmol) in ethanol (10 mL) was added 10% palladium-carboncatalyst (containing 52.8% water, 250 mg), and the suspension wasstirred in an oil bath at 40° C. for 1.5 hours in hydrogen gasatmosphere. After removing the catalyst by filtration, the filtrate wasconcentrated under reduced pressure to obtain 169 mg (98%) of the crudetarget compound as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.44 (9H, s), 1.47 (3H, s), 2.16-2.30(1H, m), 2.85 (1H, d, J=11.5 Hz), 3.01 (1H, dd, J=11.3, 7.4 Hz), 3.20(1H, dd, J=11.3, 8.6 Hz), 3.29 (1H, d, J=11.8 Hz), 4.49-4.69 (2H, m),4.98 (1H, s).

MS (ESI) m/z: 233 (M+H)⁺.

Example 247-[(3R,4R)-3-Amino-4-fluoromethyl-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3R,4R)-3-(tert-butoxycarbonylamino)-4-fluoromethyl-3-methylpyrrolidine(169 mg, 0.728 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroborane complex (268 mg, 0.742 mmol) and triethylamine(0.124 mL, 0.891 mmol) were dissolved in dimethyl sulfoxide (2 mL), andthe solution was stirred in an oil bath at 35° C. for 16 hours. Afterconcentrating the reaction mixture, a mixed solution of ethanol andwater (Ethanol:water, 9:1) (55 mL) and triethylamine (1 mL) were added,and the mixture was heated under reflux for 2 hours. After concentratingthe reaction mixture under reduced pressure, the residue was dissolvedin ethyl acetate (100 mL×2) and washed with 10% aqueous solution ofcitric acid (100 mL), water (100 m×3) and saturated aqueous solution ofsodium chloride (100 mL). The organic layer was dried with anhydroussodium sulfate, and after filtration, the filtrate was concentratedunder reduced pressure. The concentrate was dissolved in concentratedhydrochloric acid (10 mL) in an ice bath, and the mixture was stirred atroom temperature for 30 minutes and washed with chloroform (100 mL×4).To the aqueous layer was added 10 mol/l aqueous solution of sodiumhydroxide to pH 12 in an ice bath, and hydrochloric acid was added toadjust the pH to 7.4. The solution was extracted with chloroform (150mL×3), and the organic layer was dried with anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure,and the concentrate was purified by recrystallization from ethanol. Thecrystals were dried under reduced pressure to obtain 216 mg (67%) of thetitle compound as a pale yellow powder.

mp: 185-188° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.38 (3H, s), 1.50-1.62 (1H, m)1.61-1.70 (1H, m), 2.45-2.58 (1H, m), 3.39 (1H, d, J=10.5 Hz), 3.59 (3H,s), 3.70 (1H, t, J=9.3 Hz), 3.75-3.84 (2H, m), 4.02-4.09 (1H, m), 4.70(1H, ddd, J=40.7, 9.6, 6.1 Hz), 4.78-4.81 (1H, m), 4.94 (1H, ddd,J=56.6, 9.1, 6.1 Hz), 7.67 (1H, d, J=14.7 Hz), 8.48 (1H, s).

Elementary analysis for C₂₀H₂₂F₃N₃O₄.1H₂O:

Calculated: C, 54.17; H, 5.46; F, 12.85; N, 9.48

Found: C, 54.20; H, 5.52; F, 12.25; N, 8.99.

IR (ATR) ν: 2970, 2868, 1724, 1616, 1574, 1512, 1437, 1390, 1354, 1317,1298, 1271, 1192, 1142 cm⁻¹.

Reference Example 81 tert-Butyl(3S)-4-fluoro-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(Isomer A)

To a solution of tert-butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylates(6.83 g, 22.5 mmol) in tetrahydrofuran (135 mL), lithiumbistrimethylsilyl amide (27.0 mL, 27.0 mmol, 1.0M solution intetrahydrofuran) was added at 0° C., and the mixture was stirred at thesame temperature for 15 minutes. To the reaction mixture,N-fluorobenzenesulfoneimide (13.3 g, 42.2 mmol) was added at the sametemperature, and the mixture was stirred for 2 hours. Saturated aqueoussolution of ammonium chloride (300 mL) was added to the reactionmixture, and the mixture was extracted with ethyl acetate (300 mL×2).The organic layer was washed with saturated aqueous solution of sodiumchloride (300 mL), and dried with anhydrous sodium sulfate. After thefiltration, the filtrate was concentrated under reduced pressure, andthe residue was purified by silica gel column chromatography (elusion byhexane:ethyl acetate, 5:1→2:3) to obtain 5.80 g (80%) of the titlecompound (isomer A) as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.35 (3H, d, J=2.9 Hz), 1.37 (9H, s),1.56 (3H, d, J=7.1 Hz), 3.10 (1H dd, J=10.2, 1.1 Hz), 3.17 (1H, d,J=10.3 Hz), 5.23 (1H, d J=51.7 Hz), 5.50 (1H, q, J=7.1 Hz), 7.26-7.39(5H, m).

MS (ESI) m/z: 322 (M+H)⁺.

Reference Example 82 tert-Butyl(3S)-4-fluoro-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(Isomer B)

To a solution of tert-butyl(3S)-4-fluoro-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylateisomer A (3.85 g, 12.0 mmol) in tetrahydrofuran (70 mL), lithiumdiisopropyl amide (6.66 mL, 12.0 mmol, 1.8M solution in tetrahydrofuran)was added at −78° C., and the mixture was stirred at the sametemperature for 15 minutes. At the same temperature, 2,6-di-tert-butylphenol (2.97 g, 14.4 mmol) was added to the reaction mixture, and thetemperature was gradually increased with stirring in 2 hours. Saturatedaqueous solution of ammonium chloride (200 mL) was added to the reactionmixture, and the mixture was extracted with ethyl acetate (200 mL×2).The organic layer was washed with saturated aqueous solution of sodiumchloride (200 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe concentrate was purified by silica gel column chromatography(elusion by hexane ethyl acetate, 5:1→2:3) to obtain 2.32 g (60%) of thetitle compound (isomer B having a polarity higher than isomer A) as awhite solid. 1.53 g (40%) of isomer A was also recovered.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.38 (12E, s), 1.53 (3H, d, J=7.1 Hz),2.97 (1H, d, J=10.5 Hz), 3.52 (1H, d, J=10.3 Hz), 4.68 (1H, d, J=51.7Hz), 5.49 (1H, q, J=7.1 Hz), 7.26-7.39 (5H, m).

MS (ESI) m/z: 322 (M+H)⁺.

Reference Example 83 tert-Butyl(3S)-4-fluoro-3-methyl-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(Isomer A)

To a solution of tert-butyl(3S)-4-fluoro-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(isomer A) (2.58 g, 8.03 mmol) in tetrahydrofuran (50 mL) was added1.01M solution of borane in tetrahydrofuran (26.2 mL, 26.5 mmol) in anice bath and the mixture was stirred at room temperature for 15 hours.The reaction mixture was concentrated under reduced pressure, and water(5 mL), ethanol (45 ml), and triethylamine (3 mL) were added in an icebath, and the mixture was heated under reflux for 1.5 hours. Afterconcentrating the reaction mixture under reduced pressure, water (100mL) was added, and the mixture was extracted with chloroform (200 mL×2).The organic layer was washed with saturated aqueous solution of sodiumchloride (100 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure, andthe concentrate was purified by silica gel column chromatography(elusion by hexane ethyl acetate, 12:1→2:1) to obtain 2.15 g (87%) ofthe title compound (isomer A) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.29 (3H, d, J=3.7 Hz), 1.32 (3H, d,J=6.6 Hz), 1.46 (9H, s), 2.44 (H, d, J=8.8 Hz), 2.68 (1H, dd, J=31.7,11.9 Hz), 2.96 (1H, dq, J=31.0, 5.7 Hz), 3.06 (1H, d, J=8.8 Hz), 3.32(1H, q, J=6.5 Hz), 5.22 (1H, dd, J=55.2, 4.9 Hz), 7.14-7.31 (5H, m).

MS (ESI) m/z: 308 (M+H)⁺.

Reference Example 84 tert-Butyl(3S)-4-fluoro-3-methyl-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(Isomer B)

To a solution of tert-butyl(3S)-4-fluoro-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(isomer B) (1.64 g, 5.10 mmol) in tetrahydrofuran (30 mL), 1.01Msolution of borane in tetrahydrofuran (16.7 mL, 16.9 mmol) was added inan ice bath, and the mixture was stirred at room temperature for 15hours. After concentrating the reaction mixture under reduced pressure,water (5 mL), ethanol (45 mL), and triethylamine (2 mL) were added in anice bath, and the mixture was heated under reflux for 1.5 hours. Afterconcentrating the reaction mixture under reduced pressure, water (100mL) was added, and the mixture was extracted with chloroform (200 mL×2).The organic layer was washed with saturated aqueous solution of sodiumchloride (100 mL), and dried with anhydrous sodium sulfate. After thefiltration, the filtrate was concentrated under reduced pressure, andthe concentrate was purified by silica gel column chromatography(elusion by hexane ethyl acetate, 12:1→2:1) to obtain 1.55 g (99%) ofthe title compound (isomer B) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.29 (3H, d, J=1.2 Hz), 1.34 (3H, d,J=6.6 Hz), 1.43 (9H, s), 2.39 (1H, d, J=9.5 Hz), 2.74 (1H, ddd, J=34.4,12.0, 1.7 Hz), 3.02 (1H, d, J=9.5 Hz), 3.35 (1H, ddd, J=31.5, 12.2, 4.6Hz), 3.44 (1H, q, J=6.6 Hz), 4.78 (1H, ddd, J=54.2, 4.8, 1.6 Hz),7.19-7.32 (5H, m).

MS (ESI) m/z: 308 (M+H)⁺.

Reference Example 85 tert-Butyl(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylate(Isomer A)

To a solution of tert-butyl(3S)-4-fluoro-3-methyl-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylate(isomer A) (2.15 g, 6.99 mmol) in dichloromethane (40 mL),benzyloxycarbonyl chloride (1.50 mL, 10.5 mmol) was added, and themixture was stirred in an oil bath at 60° C. for 20 hours. Afterconcentrating the reaction mixture under reduced pressure, theconcentrate was purified by silica gel column chromatography (elusion byhexane:ethyl acetate, 12:1→2:1) to obtain 1.81 g (77%) of the titlecompound (isomer A) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.35 (3H, dd, J=7.8, 2.2 Hz), 1.41 (9H,br s), 3.24 (1H, dd, J=17.0, 10.9 Hz), 3.61-3.75 (2H, m), 4.08 (1H, dd,J=10.7, 2.7 Hz), 5.14 (2H, d, J=4.6 Hz), 5.17 (1H, d, J=51.0 Hz),7.26-7.38 (5H, m).

Reference Example 86 tert-Butyl(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylate(Isomer B)

To a solution of tert-butyl(3S)-4-fluoro-3-methyl-1-[(1R)-1-phenylethyl]-pyrrolidine-3-carboxylateisomer B (1.55 g, 5.04 mmol) in dichloromethane (30 mL),benzyloxycarbonyl chloride (1.08 mL, 7.56 mmol) was added, and themixture was stirred in an oil bath at 60° C. for 24 hours. Afterconcentrating the reaction mixture under reduced pressure, the residuewas purified by silica gel column chromatography (elusion byhexane:ethyl acetate, 20:1→3:2) to obtain 1.38 g (81%) of the titlecompound (isomer B) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.24 (3H, s), 1.47 (9H, s), 3.49 (1H, dd,J=26.9, 11.0 Hz), 3.60-3.80 (2H, m), 3.90 (1H, dd, J=11.0, 3.7 Hz), 4.90(1H, dd, J=51.8, 2.9 Hz), 5.14 (2H, dd, J=16.6, 11.7 Hz), 7.22-7.38 (5H,m).

MS (ESI) m/z: 360 (M+Na)⁺.

Reference Example 87(3S)-1-Benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylic Acid(Isomer A)

To a solution of tert-butyl(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylate(isomer A) (1.80 g, 5.33 mmol) in dichloromethane (10 mL),trifluoroacetic acid (10 mL) was added dropwise and, and the mixture wasstirred for 2 hours. The reaction mixture was concentrated under reducedpressure, and saturated aqueous solution of sodium hydrogencarbonate (30mL) was added to the concentrate under reduced pressure. The aqueoussolution was washed with diethylether (50 mL), and to the aqueous layerwas added 1 mol/l hydrochloric acid to pH 2 to 3, and the mixture wasextracted with chloroform (200 mL×2). The organic layer was dried withanhydrous sodium sulfate, and after filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled by adding toluene (20 mL), and dried under reduced pressure toobtain 1.86 g (quantitative) of the title compound (isomer A) as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.44 (3H, d, J=4.9 Hz), 3.32 (1H, t,J=12.0 Hz), 3.67-3.83 (2H, m), 4.14 (1H, t, J=10.3 Hz), 5.09-5.17 (2H,m), 5.22 (1H, d, J=43.9 Hz), 7.27-7.38 (5H, m).

MS (ESI) m/z: 304 (M+Na)⁺.

Reference Example 88(3S)-1-Benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylic Acid(Isomer B)

To a solution of tert-butyl(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylateisomer B (1.35 g, 4.00 mmol) in dichloromethane (7 mL), trifluoroaceticacid (7 mL) was added dropwise in an ice bath, and the mixture wasstirred for 2 hours. After concentrating the reaction mixture underreduced pressure, saturated aqueous solution of sodium hydrogencarbonate(30 mL) was added to the concentrate in an ice bath, and the aqueoussolution was washed with diethylether (50 mL). To the aqueous layer wasadded 1 mol/l hydrochloric acid to pH 2 to 3, and the solution wasextracted with chloroform (150 mL×2). The organic layer was dried withanhydrous sodium sulfate, and after filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled by adding toluene (20 mL), and dried under reduced pressure toobtain 1.25 g (quantitative) of the title compound (isomer B) as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.33 (3H, s), 3.57 (1H, dd, J=29.4, 11.0Hz), 3.66-3.86 (2H, m), 3.95 (1H, dd, J=10.8, 6.1 Hz), 4.99 (1H, dd,J=51.5, 3.2 Hz), 5.16 (2H, s), 7.15-7.52 (5H, m)

MS (ESI) m/z: 304 (M+Na)⁺.

Reference Example 89(3R)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine(Isomer A)

To a solution of(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylic acid(isomer A) (1.86 g, 5.33 mmol) in acetonitrile (40 mL), 1,1′-carbonylbis-1H-imidazole (1.30 g, 8.00 mmol) was added, and the mixture wasstirred for 1 hour. Ammonia gas was bubbled into the reaction mixturefor 1.5 hours, and the solution was concentrated under reduced pressure.Water (50 mL) was added to the concentrate, and the mixture wasextracted with chloroform (100 mL×2). The organic layer was washed withsaturated aqueous solution of sodium chloride (100 mL), and dried withanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled by adding toluene (20 mL), and dried under reduced pressure toobtain 1.80 g (quantitative) of the crude product (isomer A) in the formof amide as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.41 (3H, s), 3.40 (1H, d, J=12.0 Hz),3.60-3.97 (3H, m), 5.12-5.31 (3H, m), 5.48-5.89 (2H, m), 7.23-7.35 (5H,m).

To a solution of the crude product in the form of an amide (1.80 g, 5.33mmol) in tert-butyl alcohol (20 mL), lead tetraacetate (4.73 g, 10.7mmol) was added, and the mixture was stirred in an oil bath at 80° C.for 15 minutes. After allowing to cool, sodium hydrogencarbonate (5 g)and diethylether (20 mL) were added to the reaction mixture, and themixture was stirred for 30 minutes in an ice bath. After removing theinsoluble content by filtration through celite, the filtrate and thesolution used for the washing were combined, and washed with saturatedaqueous solution of sodium hydrogencarbonate (50 mL) and saturatedaqueous solution of sodium chloride (50 mL). The organic layer was driedwith anhydrous magnesium sulfate, and after filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was purified bysilica gel column chromatography (elusion by hexane:ethyl acetate,20:1→3:2) to obtain 1.00 g (53%) of the title compound (isomer A) as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.42 (9H, s), 1.51 (3H, dd, J=8.8, 2.9 Hz),3.42 (1H, dd, J=11.8, 2.5 Hz), 3.53 (1H, d, J=11.5 Hz), 3.59-3.72 (1H,m), 3.71-3.87 (1H, m), 4.50 (1H, d, J=28.7 Hz), 5.14 (2H, s), 5.35 (1H,dd, J=52.0, 26.0 Hz), 7.26-7.39 (5H, m).

MS (ESI) m/z: 375 (M+Na)⁺.

Reference Example 90(3R)-1-Benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine(Isomer B)

To a solution of(3S)-1-benzyloxycarbonyl-4-fluoro-3-methylpyrrolidine-3-carboxylic acid(isomer B) (1.25 g, 4.00 mmol) in acetonitrile (40 mL), 1,1′-carbonylbis-1H-imidazole (973 mg, 6.00 mmol) was added, and the mixture wasstirred for 1 hour. After bubbling ammonia gas in the reaction mixturefor 1.5 hours, the mixture was concentrated under reduced pressure.Water (50 mL) was added to the concentrate, and the solution wasextracted with chloroform (100 mL×2). The organic layer was washed withsaturated aqueous solution of sodium chloride (100 mL), and dried withanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled with toluene (20 mL), and dried under reduced pressure toobtain 1.20 g (quantitative) of the crude product in the form of anamide (isomer B) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.31 (3H, s), 3.61 (1H, dd, J=25.6, 10.4Hz), 3.69-3.79 (1H, m), 3.83 (2H, dd, J=16.7, 3.9 Hz), 4.96 (1H, d,J=51.5 Hz), 5.15 (2H, br s), 5.58 (2H, d, J=59.6 Hz), 7.23-7.40 (5H, m).

To a solution of the crude product in the form of an amide (1.20 g, 4.00mmol) in tert-butyl alcohol (15 mL) was added lead tetraacetate (3.55 g,8.00 mmol), and the mixture was stirred in an oil bath at 80° C. for 1hour. After allowing the reaction mixture to cool, sodiumhydrogencarbonate (4 g) and diethylether (20 mL) were added to thereaction mixture, and the mixture was stirred in an ice bath for 1 hour.The insoluble content was removed by filtration through celite, and thefiltrate and the washing solution of the insoluble content were combinedand washed with saturated aqueous solution of sodium hydrogencarbonate(50 mL) and saturated aqueous solution of sodium chloride (50 mL). Theorganic layer was dried with anhydrous magnesium sulfate, and thesolvent was removed by distillation under reduced pressure. The residuewas purified by silica gel column chromatography (elusion withhexane:ethyl acetate, 20:1→3:2) to obtain 1.03 g (73%) of the titlecompound (isomer B) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.42 (3H, dd, J=4.4, 1.2 Hz), 1.44-1.45(9H, m), 3.42 (1H, t, J=11.2 Hz), 3.62-3.71 (1H, m), 3.76 (1H, dd,J=10.3, 2.5 Hz), 3.84 (1H, t, J=10.7 Hz), 4.84 (1H, d, J=54.4 Hz), 4.92(1H, d, J=56.9 Hz), 5.14 (2H, dd, J=15.3, 13.1 Hz), 7.26-7.52 (5H, m).

MS (ESI) m/z: 375 (M+Na)⁺.

Reference Example 91(3R)-3-(tert-Butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine (IsomerA)

To a solution of(3R)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine(isomer A) (271 mg, 0.769 mmol) in ethanol (10 mL) was added 10%palladium-carbon catalyst (containing 52.8% water, 27.0 mg), and thesuspension was stirred for 2 hours in hydrogen gas atmosphere. Afterremoving the catalyst by filtration, the filtrate was concentrated underreduced pressure to obtain 156 mg (93%) of the crude target compound(isomer A) as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.43-1.44 (12H, m), 3.06 (1H, dd, J=13.4,1.7 Hz), 3.13 (1H, d, J=13.4 Hz), 3.34 (1H, dd, J=13.4, 4.6 Hz), 3.42(1H, dd, J=13.4, 4.6 Hz), 4.58 (1H, s), 5.16 (1H, d, J=53.7 Hz).

MS (ESI) m/z: 219 (M+H)⁺.

Reference Example 92(3R)-3-(tert-Butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine (IsomerB)

To a solution of(3R)-1-benzyloxycarbonyl-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine(isomer B) (304 mg, 0.863 mmol) in ethanol (12 mL) was added 10%palladium-carbon catalyst (containing 52.8% water, 30.0 mg), and thesuspension was stirred for 2 hours in hydrogen gas atmosphere. Afterremoving the catalyst by filtration, the filtrate was concentrated underreduced pressure to obtain 182 mg (97%) of the crude target compound(isomer B) as a colorless transparent syrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.40 (3H, d, J=1.7 Hz), 1.45 (9H, s),3.11-3.17 (2H, m), 3.19-3.25 (1H, m), 3.31 (1H, dd, J=13.7, 4.4 Hz),4.75 (1H, dd, J=55.3, 3.6 Hz), 4.99 (1H, s).

MS (ESI) m/z: 219 (M+H)⁺.

Example 257-[(3R)-3-Amino-4-fluoro-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid

(3R)-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine isomer A(156 mg, 0.713 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (278 mg, 0.769 mmol), and triethylamine(0.129 mL, 0.923 mmol) were added to dimethyl sulfoxide (2 mL) and themixture was stirred in an oil bath at 35° C. for 19 hours. Afterconcentrating the mixture, a mixed solution of ethanol and water(ethanol:water, 9:1) (60 mL) and triethylamine (2 mL) were added to theconcentrate, and the mixture was heated under reflux for 2 hours. Thereaction mixture was concentrated under reduced pressure, and theconcentrate was dissolved in ethyl acetate (100 mL×2). The solution waswashed with 10% aqueous solution of citric acid (100 mL), water (100mL×3), and saturated aqueous solution of sodium chloride (100 mL). Theorganic layer was dried with anhydrous sodium sulfate, and afterfiltration, the filtrate was concentrated under reduced pressure. Theconcentrate was dissolved in concentrated hydrochloric acid (10 mL) inan ice bath, and the mixture was stirred at room temperature for 30minutes. The reaction mixture was washed with chloroform (100 mL×4), andto the aqueous layer was added 10 mol/l aqueous solution of sodiumhydroxide to pH 12 in an ice bath, and hydrochloric acid was added toadjust the pH to 7.4. The solution was extracted with chloroform (150mL×3), and the organic layer was dried with anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure,and the concentrate was purified by recrystallization from ethanol. Thecrystals were dried to obtain 165 mg (52%) of the title compound (fromisomer A of substituent at position 7) as a pale yellow powder.

mp: 160-163° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.38 (3H, d, J=2.5 Hz), 1.40-1.48(1H, m), 1.50-1.62 (1H, m), 3.44 (1H, d, J=10.3 Hz), 3.59 (3H, s),3.68-3.80 (2H, m), 4.00-4.05 (1H, m), 4.29 (1H, dd, J=40.0, 13.5 Hz),4.82-4.87 (1H, m), 5.03 (1H, dd, J=65.7, 4.7 Hz), 7.69 (1H, d, J=14.2Hz), 8.41 (1H, d, J=2.9 Hz)

Elementary analysis for C₁₉H₂₀F₃N₃O₄.25H₂O:

Calculated: C, 54.87; H, 4.97; F, 13.70; N, 10.10.

Found: C, 54.71; H, 4.98; F, 13.54; N, 10.09.

IR (ATR) ν: 3386, 2972, 2881, 1722, 1624, 1518, 1452, 1373, 1325, 1279,1223, 1190, 1149, 1122 cm⁻¹.

Example 267-[(3R)-3-Amino-4-fluoro-3-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3R)-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine isomer B(182 mg, 0.833 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (311 mg, 0.863 mmol), and triethylamine(0.144 mL, 1.04 mmol) were dissolved in dimethyl sulfoxide (2 mL), andthe mixture was stirred in an oil bath at 35° C. for 19 hours. Afterconcentrating the mixture, a mixed solution of ethanol and water(ethanol:water, 9:1) (60 mL) and triethylamine (3 mL) were added, an themixture was heated under reflux for 2 hours. The reaction mixture wasconcentrated under reduced pressure, and the concentrate was dissolvedin ethyl acetate (100 mL×2), and the solution was washed with 10%aqueous solution of citric acid (100 mL), water (100 m×3), and saturatedaqueous solution of sodium chloride (100 mL). The organic layer wasdried with anhydrous sodium sulfate, and after filtration, the filtratewas concentrated under reduced pressure. The concentrate was dissolvedin concentrated hydrochloric acid (10 mL), and the mixture was stirredat room temperature for 30 minutes. The reaction mixture washed withchloroform (100 mL×4), and to the aqueous layer was added 10 mol/laqueous solution of sodium hydroxide to pH 12 in an ice bath, andhydrochloric acid was added to the solution to adjust the pH to 7.4. Thesolution was extracted with chloroform (150 mL×3), and the organic layerwas dried with anhydrous sodium sulfate. After filtration, the filtratewas concentrated under reduced pressure. The concentrate was purified byrecrystallization from ethanol, and the crystals were dried underreduced pressure to obtain 176 mg (51%) of the title compound (fromisomer B of substituent at position 7) as a pale yellow powder.

mp: 206-208° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.29 (3H, br s), 1.57-1.74 (2H, m),3.37 (1H, d, J=9.8 Hz), 3.56-3.67 (1H, m), 3.60 (3H, s), 3.83 (1H, d,J=10.0 Hz), 4.08 (1H, q, J=6.3 Hz), 4.34 (1H, dd, J=43.3, 13.1 Hz),4.77-4.84 (1H, m), 4.93 (1H, ddd, J=52.7, 10.0, 3.2 Hz), 7.68 (1H, d,J=14.5 Hz), 8.50 (1H, s). Elementary analysis for C₁₉H₂₀F₃N₃O₄:

Calculated: C, 55.47; H, 4.90; F, 13.85; N, 10.21.

Found: C, 55.29; H, 4.86; F, 13.99; N, 10.33.

IR (ATR) ν: 3373, 3300, 3074, 2979, 2881, 2837, 1709, 1620, 1510, 1435,1378, 1338, 1313, 1269, 1225, 1186, 1130 cm⁻¹.

Example 277-[(3R)-3-Amino-4-fluoro-3-methylpyrrolidine-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3R)-3-(tert-butoxycarbonylamino)-4-fluoro-3-methylpyrrolidine isomer A(397 mg, 1.82 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (418 mg, 1.21 mmol), and triethylamine (0.202mL, 1.45 mmol) were dissolved in sulfolane (2 mL) in an oil bath at 35°C., and the mixture was stirred for 264 hours. After concentrating themixture, a mixed solution of ethanol and water (ethanol:water, 9:1) (80mL) and triethylamine (1 mL) were added to the concentrate in an oilbath at 90° C., and the mixture was stirred for 30 minutes. Afterconcentrating the reaction mixture under reduced pressure, the residuewas dissolved in ethyl acetate (200 mL×2), and the solution was washedwith 10% aqueous solution of citric acid (100 mL), water (100 mL×3), andsaturated aqueous solution of sodium chloride (100 mL). The organiclayer was dried with anhydrous sodium sulfate, and after filtration, thefiltrate was concentrated under reduced pressure, and the residue waspurified by short silica gel column chromatography (elusion bychloroform methanol, 49:1→9:1). The residue was dissolved inhydrochloric acid (20 mL), and the solution was stirred at roomtemperature for 30 minutes, and the washed with chloroform (100 mL×5).To the aqueous layer, 10 mol/l aqueous solution of sodium hydroxide wasadded to pH 12 in an ice bath, and hydrochloric acid was added to adjustthe pH to 7.4. The solution was extracted with chloroform (150 mL×4),and the organic layer was dried with anhydrous sodium sulfate. After thefiltration, the filtrate was concentrated under reduced pressure, andthe concentrate was purified by recrystallization from ethanol, and thecrystals were dried under reduced pressure to obtain 28.3 mg (6%) of thetitle compound (from isomer A of substituent at position 7) as a paleyellow powder.

mp: 215-217° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.20-1.32 (1H, m), 1.37 (3H, d, J=2.7Hz), 1.54-1.67 (1H, m), 2.51 (3H, s), 3.17 (1H, d, J=9.8 Hz), 3.47 (1H,dd, J=29.0, 12.6 Hz), 3.86 (1H, d, J=10.0 Hz), 4.05-4.12 (1H, m), 4.30(1H, ddd, J=40.0, 13.2, 3.2 Hz), 4.80-4.85 (1H, m), 5.01 (1H, ddd,J=67.8, 9.4, 4.6 Hz), 7.70 (1H, d, J=14.0 Hz), 8.45 (1H, d, J=3.4 Hz).

Elementary analysis for C₁₉H₂₀F₃N₃O₃0.75H₂O:

Calculated: C, 55.81; H, 5.30; N, 10.28.

Found: C, 55.81; H, 4.89; N, 10.14.

IR (ATR) ν: 3394, 3097, 2970, 2941, 2870, 1726, 1618, 1599, 1508, 1456,1425, 1319, 1267, 1225, 1190, 1146 cm⁻¹.

Reference Example 93 tert-Butyl(3S)-3-hydroxymethyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate (4.00 g, 13.8mmol) in N,N-dimethylformamide (40 mL), paraformaldehyde (0.830 g, 27.7mmol) and sodium hydride (0.600 g, 55% in oil, 13.8 mmol) were added atroom temperature, and the mixture was stirred at the same temperaturefor 30 minutes. To the reaction mixture, 10% aqueous solution of citricacid (150 mL) was added in an ice bath, and the solution was extractedwith ethyl acetate (300 mL×2). The organic layer was washed with water(100 mL×2) and saturated aqueous solution of sodium chloride (100 mL),and dried with anhydrous sodium sulfate. After the filtration, thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (elusion by hexane:ethylacetate, 7:3→1:4) to obtain 1.03 g (23%) of the title compound as awhite solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.35 (9H, s), 1.53 (3H, d, J=7.3 Hz),2.40 (1H, d, J=17.3 Hz), 2.51 (1H, dd, J=7.8, 5.4 Hz), 2.78 (1H, d,J=17.1 Hz), 3.21 (1H, d, J=10.3 Hz), 3.39 (1H, d, J=10.5 Hz), 3.61 (1H,dd, J=11.2, 7.8 Hz), 3.77 (1H, dd, J=11.2, 5.4 Hz), 5.51 (1H, q, J=7.2Hz), 7.26-7.37 (5H, m)

MS (ESI) m/z: 320 (M+H)⁺.

Reference Example 94 tert-Butyl (3S)-3-{[tert-butyl(dimethyl)silyloxy]methyl}-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl(3S)-3-hydroxymethyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(8.23 g, 25.8 mmol) and imidazole (2.63 g, 38.7 mmol) inN,N-dimethylformamide (150 mL), tert-butyl dimethylsilyl chloride (4.66g, 31.0 mmol) was added in an ice bath, and the mixture was stirred atroom temperature for 4 hours. To the reaction mixture, saturated aqueoussolution of ammonium chloride (300 mL) was added in an ice bath, and themixture was extracted with diethylether (300 mL×2). The organic layerwas washed with water (300 mL×2) and saturated aqueous solution ofsodium chloride (200 mL), and dried with anhydrous magnesium sulfate.After filtration, the filtrate was concentrated under reduced pressure,and the residue was purified by silica gel column chromatography(elusion by hexane:ethyl acetate, 10:1→1:1) to obtain 7.98 g (71%) ofthe title compound as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.05 (3H, s), 0.06 (3H, s), 0.88 (9H, s),1.35 (9H, s), 1.51 (3H, d, J=7.1 Hz), 2.47 (1H, d, J=17.1 Hz), 2.77 (1H,d, J=17.3 Hz), 3.28 (2H, dd, J=26.7, 10.1 Hz), 3.68 (2H, dd, J=14.4, 9.5Hz), 5.49 (1H, q, J=7.1 Hz), 7.25-7.35 (5H, m).

Reference Example 95 tert-Butyl (3S)-3-{[tert-butyl(dimethyl)silyloxy]methyl}-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a solution of tert-butyl (3S)-3-{[tert-butyl(dimethyl)silyloxy]methyl}-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(8.65 g, 19.9 mmol) and iodomethane (1.37 mL, 21.9 mmol) intetrahydrofuran (173 mL), lithium bistrimethylsilyl amide (21.9 mL, 21.9mmol, 1M solution in tetrahydrofuran) was added in an ice bath, and themixture was stirred at the same temperature for 30 minutes. To thereaction mixture, saturated aqueous solution of ammonium chloride (300mL, was added, and the mixture was extracted with ethyl acetate (300mL×2). The organic layer was washed with saturated aqueous solution ofsodium chloride (200 mL), and dried with anhydrous sodium sulfate. Afterthe filtration, the filtrate was concentrated under reduced pressure,and the residue was purified by silica gel column chromatography(elusion by hexane:ethyl acetate, 7:1→1:1) to obtain 3.72 g (42%) of thetitle compound (single component) as a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.05 (3H, s), 0.06 (3H, s), 0.88 (9H, s),1.12 (3H, d, J=7.3 Hz), 1.38 (9H, s), 1.51 (3H, d, J=7.1 Hz), 2.31 (1H,q, J=7.4 Hz), 3.29 (11H, d, J=10.3 Hz), 3.39 (1H, d, J=10.3 Hz), 3.50(1H, dd, J=9.4, 6.0 Hz), 3.83 (1H, d, J=9.5 Hz), 5.47 (1H, q, J=7.2 Hz),7.24-7.36 (5H, m)

MS (ESI) m/z: 448 (M+H)⁺.

Reference Example 96 tert-Butyl(3S)-3-hydroxymethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

Tert-butyl (3S)-3-{[tert-butyl(dimethyl)silyloxy]methyl}-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(3.72 g, 8.31 mmol) was dissolved in tetrahydrofuran (70 mL), andtetrabutylammonium fluoride (12.5 mL, 1.0 mol/l solution intetrahydrofuran, 12.5 mmol) was added dropwise in an ice bath. Themixture was stirred at the same temperature for 1 hour. Afterconcentrating the mixture, saturated aqueous solution of ammoniumchloride (200 mL) was added, and the mixture was extracted with ethylacetate (200 mL×2). The organic layer was washed with saturated aqueoussolution of sodium chloride (100 mL), and dried with anhydrous sodiumsulfate. After filtration, the filtrate was concentrated under reducedpressure. The concentrate was purified by silica gel columnchromatography (elusion by hexane:ethyl acetate=3:1→1:2) to obtain 1.87g (67%) of the title compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.18 (3H, d, J=7.6 Hz), 1.32 (9H, s),1.52 (3H, d, J=7.1 Hz), 2.43-2.49 (2H, m), 3.30 (2H, dd, J=19.0, 10.5Hz), 3.57 (1H, dd, J=11.1, 6.7 Hz), 3.89 (1H, dd, J=11.0, 5.9 Hz), 5.48(1H, q, J=7.2 Hz), 7.27-7.36 (5H, m).

MS (ESI) M/Z: 334 (M+H)⁺.

Reference Example 97 tert-Butyl(3R)-3-fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

Tert-butyl(3S)-3-hydroxymethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(1.70 g, 5.10 mmol) was dissolved in dichloromethane (20 mL). To thissolution, toluene (20 mL) was added, and diethylaminosulfur trifluoride(1.68 mL, 12.8 mmol) was added dropwise in an ice bath. After stirringat 60° C. for 8 hours, saturated aqueous solution of sodiumhydrogencarbonate (50 mL) was added to the reaction mixture, and themixture was extracted by ethyl acetate (100 mL×2). The organic layer waswashed with saturated aqueous solution of sodium chloride (100 mL), anddried with anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was then purifiedby silica gel column chromatography (solution by hexane:ethyl acetate,9:1→2:3) to obtain 0.910 mg (53%) of the title compound as a pale yellowoily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.17 (3H, d, J=7.6 Hz), 1.37 (9H, d,J=0.5 Hz), 1.54 (3H, d, J=7.1 Hz), 2.35-2.42 (1H, m), 3.37 (2H, t,J=12.1 Hz), 4.33 (1H, dd, J=46.9, 9.2 Hz), 4.65 (1H, dd, J=46.8, 9.1Hz), 5.48 (1H, q, J=7.2 Hz), 7.25-7.37 (5H, m) MS (ESI) m/z: 336 (M+H)⁺.

Reference Example 98(3R)-3-Fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylicAcid

To a solution of tert-butyl(3R)-3-fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate(910 mg, 2.71 mmol) in dichloromethane (9 mL), trifluoroacetic acid (9mL) was added dropwise in an ice bath, and the mixture was stirred atroom temperature for 3 hours. After concentrating the reaction mixtureunder reduced pressure, saturated aqueous solution of sodiumhydrogencarbonate (20 mL) was added to the concentrate in an ice bath,and the aqueous solution was washed with diethylether (50 mL). To theaqueous layer was added 1 mol/l hydrochloric acid to pH 2 to 3 in an icebath, and the solution was extracted with chloroform (100 mL×2). Theorganic layer was washed with saturated aqueous solution of sodiumchloride (100 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure. Theconcentrate was azeotropically distilled by adding toluene (20 mL), anddried under reduced pressure to obtain 910 mg (quantitative) of thetitle compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.18 (3H, d, J=7.4 Hz), 1.55 (3H, d,J=7.1 Hz), 2.49 (1H, q, J=7.4 Hz), 3.35-3.50 (2H, m), 4.38 (1H, dd,J=46.7, 9.2 Hz), 4.71 (1H, dd, J=46.8, 9.3 Hz), 5.50 (1H, q, J=7.1 Hz),7.26-7.37 (5H, m).

Reference Example 99(3S)-3-(tert-Butoxycarbonylamino)-3-fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine

To a solution of(3R)-3-fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylicacid (910 mg, 2.71 mmol) in acetonitrile (20 mL),1,1′-carbonylbis-1H-imidazole (659 mg, 4.07 mmol) was added, and themixture was stirred for 20 minutes. Ammonia gas was bubbled into thereaction mixture for 1.5 hours, and the solution was concentrated underreduced pressure. Water (50 mL) was added to the concentrate, and themixture was extracted with chloroform (100 mL×2). The organic layer waswashed with saturated aqueous solution of sodium chloride (100 mL) anddried with anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was azeotropicallydistilled by adding toluene (20 mL), and dried under reduced pressure toobtain 800 mg (quantitative) of the crude produce in the form of amideas a colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.18 (3H, d, J=7.4 Hz), 1.56 (3H, d,J=7.4 Hz), 2.72 (1H, q, J=7.3 Hz), 3.26-3.37 (2H, m), 4.52 (1H, dd,J=21.0, 9.7 Hz), 4.64 (1H, dd, J=20.7, 9.7 Hz), 5.15 (2H, d, J=55.4 Hz),5.57 (1H, q, J=7.0 Hz), 7.26-7.41 (5H, m).

To a solution of the crude product in the form of an amide (800 mg, 2.71mmol) in tert-butyl alcohol (10 mL), lead tetraacetate (2.40 g, 5.42mmol) was added, and the mixture was stirred in an oil bath at 80° C.for 30 minutes. After allowing to cool, sodium hydrogencarbonate (2.5 g)and diethylether (20 mL) were added to the reaction mixture, and themixture was stirred in an ice bath for 30 minutes. The insoluble contentwas removed by filtration through celite, and filtrate and the solutionused for the washing were combined and washed with saturated aqueoussolution of sodium hydrogencarbonate (50 mL) and saturated aqueoussolution of sodium chloride (50 mL). The organic layer was dried withanhydrous magnesium sulfate, and after filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was purified bysilica gel column chromatography (elusion by hexane:ethyl acetate,20:1→3:2) to obtain 485 mg (51%) of the title compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.18 (3H, d, J=7.4 Hz), 1.34 (9H, s),1.51 (3H, d, J=7.6 Hz), 2.71-2.79 (1H, m), 3.16-3.24 (1H, m), 3.39 (1H,dd, J=10.7, 1.3 Hz), 4.46 (1H, dd, J=47.3, 9.1 Hz), 4.55 (1H, s), 4.71(1H, dd, J=46.8, 9.1 Hz), 5.50 (1H, q, J=7.1 Hz), 7.23-7.34 (5H, m).

Reference Example 100(3S)-3-(tert-Butoxycarbonylamino)-3-fluoromethyl-4-methyl-1-[(1R)-1-phenylethyl]pyrrolidine

To a solution of(3S)-3-(tert-butoxycarbonylamino)-3-fluoromethyl-4-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine(485 mg, 1.38 mmol) in tetrahydrofuran (10 mL), 1.00M solution of boranein tetrahydrofuran (4.57 mL, 4.57 mmol) was added in an ice bath, andthe mixture was stirred at room temperature for 15 hours. Afterconcentrating the reaction mixture under reduced pressure, water (1 mL),ethanol (9 mL), and triethylamine (1 mL) were added in an ice bath, andthe mixture was heated under reflux for 1.5 hours. After concentratingthe reaction mixture under reduced pressure, water (100 mL) was added,and the mixture was extracted with chloroform (100 mL×2). The organiclayer was washed with saturated aqueous solution of sodium chloride (100mL), and dried with anhydrous sodium sulfate. After the filtration, thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (elusion by hexane ethylacetate, 12:1→1:1) to obtain 350 mg (75%) of the title compound as acolorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.03 (3H, d, J=6.8 Hz), 1.32 (3H, d,J=6.6 Hz), 1.41 (9H, s), 2.11 (1H, t, J=8.5 Hz), 2.19-2.29 (1H, m), 2.38(1H, dd, J=10.1, 4.5 Hz), 2.84 (1H, d, J=10.3 Hz), 3.16 (1H, t, J=8.1Hz), 3.25 (1H, q, J=6.5 Hz), 4.54 (2H, d, J=48.3 Hz), 4.65 (1H, s),7.19-7.31 (5H, m).

MS (ESI) m/z: 337 (M+H)⁺.

Reference Example 101(3S)-3-(tert-Butoxycarbonylamino)-3-fluoromethyl-4-methylpyrrolidine

To a solution of(3S)-3-(tert-butoxycarbonylamino)-3-fluoromethyl-4-methyl-1-[(1R)-1-phenylethyl]pyrrolidine(200 mg, 0.594 mmol) in ethanol (12 mL) was added 10% palladium-carboncatalyst (containing 52.8% water, 200 mg), and the suspension wasstirred in an oil bath at 40° C. for 2 hours in hydrogen gas atmosphere.After removing the catalyst by filtration, the solvent was removed bydistillation under reduced pressure to obtain 150 mg (quantitative) ofthe crude title compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.06 (3H, d, J=6.9 Hz), 1.44 (9H, s),2.28 (1H, q, J=7.4 Hz), 2.80 (1H, t, J=10.0 Hz), 3.24 (1H, d, J=12.3Hz), 3.32 (2H, dd, J=11.0, 8.1 Hz), 4.54 (2H, d, J=47.3 Hz), 4.74 (1H,s)

Example 287-[(3S)-3-Amino-3-fluoromethyl-4-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S)-3-(tert-butoxycarbonylamino)-3-fluoromethyl-4-methylpyrrolidine(150 mg, 0.594 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (214 mg, 0.594 mmol), and triethylamine(0.0994 mL, 0.713 mmol) were dissolved in dimethyl sulfoxide (2 mL), andthe mixture was stirred in an oil bath at 35° C. for 15 hours. Afterconcentrating the mixture, a mixed solution of ethanol and water(ethanol:water, 9:1) (11 mL) and triethylamine (0.5 mL) were added tothe concentrate, and the mixture was heated under reflux for 2 hours.The reaction mixture was concentrated under reduced pressure, and theconcentrate was dissolved in ethyl acetate (100 mL×2), and the solutionwas washed with 10% aqueous solution of citric acid (100 mL), watermL×3), and saturated aqueous solution of sodium chloride mL). Theorganic layer was dried with anhydrous sodium sulfate, and afterfiltration, the filtrate was concentrated under reduced pressure. Theconcentrate was dissolved in concentrated hydrochloric acid (10 mL), andafter stirring the solution at room temperature for 30 minutes, thereaction mixture was washed with chloroform (100 mL×4). To the aqueouslayer was added 10 mol/l aqueous solution of sodium hydroxide to pH 12in an ice bath, and hydrochloric acid was added to adjust the pH to 7.4.The solution was extracted with chloroform (150 mL×3), and the organiclayer was dried with anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure. The concentrate waspurified by recrystallization from ethanol, and the crystals were driedunder reduced pressure to obtain 115 mg (45%) of the title compound as apale yellow powder.

mp: 167-169° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.07 (3H, d, J=6.9 Hz), 1.37-1.50(1H, m), 1.50-1.62 (1H, m), 2.34 (1H, q, J=7.7 Hz), 3.45 (1H, d, J=11.0Hz), 3.52-3.58 (1H, m), 3.59 (3H, s), 3.82 (1H, d, J=8.6 Hz), 3.87 (1H,d, J=12.5 Hz), 4.00-4.05 (1H, m), 4.51 (2H, d, J=47.3 Hz), 5.01 (1H, d,J=65.0 Hz), 7.67 (1H, d, J=14.5 Hz), 8.41 (1H, d, J=2.7 Hz). Elementaryanalysis for C₂₀H₂₂F₃N₃O₄.0.25H₂O:

Calculated: C, 55.88; H, 5.28; F, 13.26; N, 9.77.

Found: C, 55.66; H, 5.21; F, 13.26; N, 9.97.

MS (ESI) m/z: 426 (M+H)⁺.

IR (ATR) ν: 2962, 2939, 2877, 1716, 1622, 1514, 1452, 1441, 1363, 1327,1273, 1184, 1124 cm⁻¹.

Example 297-[(7S)-7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

A mixture of(7S)-7-(tert-butoxylcarbonylamino)-7-methyl-5-azaspiro[2.4]heptane (523mg, 2.31 mmol),7-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (538 mg, 1.927 mmol), triethylamine (0.537 mL, 3.85 mmol), anddimethyl sulfoxide (6 mL) was stirred in nitrogen atmosphere in an oilbath at 75° C. for 5 days, and in an oil bath at 85° C. for 2 days. Tothe reaction mixture was added 10% aqueous solution of citric acid (10mL), and the mixture was extracted with ethyl acetate (50 mL). Theorganic layer was washed with water (10 mL×2) and saturated aqueoussolution of sodium chloride (10 mL), and dried with anhydrous sodiumsulfate. After filtration, the filtrate was concentrated under reducedpressure. The residue was purified by silica gel column chromatography(silica gel, 10 g; elusion by chloroform→chloroform:methanol, 98:2) toobtain a pale yellow foam solid. The purified pale yellow foam solid wasdissolved in concentrated hydrochloric acid (8 mL) at room temperature,and after transferring the resulting acidic aqueous solution using 6Nhydrochloric acid to a separatory funnel, solution was washed withchloroform (50 mL×8). To the aqueous layer was added 10 mol/l aqueoussolution of sodium hydroxide to pH 12.0 in an ice bath, and hydrochloricacid was added to adjust the pH to 7.4. The solution was extracted witha mixed solvent of chloroform and methanol (chloroform:methanol, 9:1)(100 mL×3), and lower layer of a mixed solvent chloroform, methanol, andwater (chloroform:methanol:water, 7:3:1) (100 mL). The organic layer wasdried with anhydrous sodium sulfate, and after filtration, the filtratewas concentrated under reduced pressure. The concentrate was purified byrecrystallization from ethanol-isopropanol system, and the crystals weredried under reduced pressure to obtain 332 mg (2 steps, 42%) of thetitle compound as a pale yellow powder.

mp: 157-159° C.

[α]_(D) ^(25.0)=−144.2° (c=0.197, 0.1N NaOE)

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.50-0.54 (1H, m), 0.58-0.62 (1H, m),0.69-0.80 (2H, m), 1.06 (3H, s), 1.22-1.33 (1H, m), 1.57-1.63 (1H, m),2.42 (3H, s), 3.14 (1H, d, J=9.8 Hz), 3.35 (1H, d, J=9.6 Hz), 3.65 (1H,d, J=9.6 Hz), 3.89 (1H, d, J=9.8 Hz), 4.04-4.09 (1H, m), 4.93-5.11 (1H,m), 7.06 (1H, d, J=9.1 Hz), 7.99 (1H, d, J=9.1 Hz), 8.45 (1H, d, J=2.9Hz)

Elementary analysis for C₂₁H₂₄FN₃O₃1.25H₂O:

Calculated: C, 61.83; H, 6.55; F, 4.66; N, 10.30.

Found: C, 61.65; H, 6.30; F, 4.77; N, 9.88.

MS (FAB) m/z: 386 (M+H)⁺.

IR (ATR) ν: 1718, 1608, 1572, 1508, 1460, 1429, 1390, 1358, 1317, 1279,1259, 1196 cm⁻¹.

Example 307-[(7S)-7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-1-cyclopropyl-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

A mixture of(7S)-7-(tert-butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane (524mg, 2.31 mmol),1-fluorocyclopropyl-7-fluoro-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid (503 mg, 1.925 mmol), triethylamine (0.537 mL, 3.85 mmol), anddimethyl sulfoxide (6 mL) was stirred in nitrogen atmosphere in an oilbath at 75° C. for 5 days, and in an oil bath at 85° C. for 2 days. Tothe reaction mixture was added 10% aqueous solution of citric acid (10mL), and the mixture was extracted with ethyl acetate (50 mL). Theorganic layer was washed with water (10 mL×2) and saturated aqueoussolution of sodium chloride (10 mL), and dried with anhydrous sodiumsulfate. After filtration, the filtrate was concentrated under reducedpressure. The residue was purified by silica gel column chromatography(silica gel, 10 g; elusion by chloroform→chloroform:methanol, 98:2) toobtain a pale yellow foam solid. The purified pale yellow foam solid wasdissolved in concentrated hydrochloric acid (8 mL) at room temperature,and after transferring the resulting acidic aqueous solution to aseparatory funnel while washing with 6N hydrochloric acid, the solutionwas washed with chloroform (50 mL×8). To the aqueous layer was added 10mol/l aqueous solution of sodium hydroxide to pH 12.0 in an ice bath,and hydrochloric acid was added to adjust the pH to 7.4. The solutionwas extracted with a mixed solvent of chloroform and methanol(chloroform:methanol, 9:1) (100 mL×3), and the organic layer was driedwith anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure. The concentrate was purified byrecrystallization from ethanol, and dried under reduced pressure toobtain 268 mg (0.641 mmol, 2 steps, 33%) of the title compound as a paleyellow powder.

mp: 227-230° C.

[α]_(D) ^(25.0)=−38.9° (c=0.211, 0.1N NaOH).

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 0.53 (2H, m), 0.72 (2H, m), 0.79 (2H,m), 1.08 (3H, s), 1.15 (1H, m), 2.41 (3H, s), 3.35 (1H, d, J=10.0 Hz),3.40 (1H, d, J=9.6 Hz), 3.49 (1H, d, J=9.8 Hz), 3.57 (1H, d, J=9.8 Hz),4.04-4.07 (1H, m), 7.01 (1H, d, J=9.1 Hz), 7.95 (1H, d, J=9.1 Hz), 8.56(1E, s)

Elementary analysis for C₂₁H₂₅N₃O₃.1.0EtOH.0.25H₂O:

Calculated: C, 66.09; H, 7.60; N, 10.05

Found: C, 66.38; H, 7.48; N, 10.26.

MS (FAB) m/z: 368 (M+H)⁺.

IR (ATR) ν: 2964, 2916, 2850, 1711, 1610, 1545, 1508, 1466, 1427, 1390,1352, 1313, 1254, 1194 cm⁻¹.

Example 317-[(3S)-3-Amino-3-fluoromethyl-4-methylpyrrolidin-1-yl]-6-fluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(3S)-3-(tert-butoxycarbonylamino)-3-fluoromethyl-4-methylpyrrolidine(155 mg, 0.663 mmol),6,7-difluoro-1-[(1R,2S)-2-fluoro-1-cyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroboron complex (228 mg, 0.661 mmol), and triethylamine(0.111 ml, 0.795 mmol) were dissolved in sulfolane (0.8 ml), and themixture was stirred in an oil bath at 35° C. for 480 hours. Afterconcentrating the reaction solution, a mixed solution of ethanol andwater (ethanol:water, 9:1) (5 ml) and triethylamine (0.5 ml) were added,and the mixture was stirred in an oil bath at 80° C. for 30 minutes. Thereaction system was concentrated under reduced pressure, and theconcentrate was dissolved in ethyl acetate (100 ml×2), and washed with10% aqueous solution of citric acid (100 ml), water (100 ml×2), andsaturated aqueous solution of sodium chloride (100 ml). The organiclayer was dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. The residue was purifiedby short silica gel column chromatography (chloroform:methanol,99:1→4:1). The residue was dissolved in concentrated hydrochloric acid(10 ml) in an ice bath, and the solution was stirred at room temperaturefor 30 minutes. The reaction solution was washed with chloroform (100ml×3), and to the aqueous layer was added 10 mol/l aqueous solution ofsodium hydroxide to pH 12 in an ice bath, and hydrochloric acid wasadded to adjust the pH to 7.4. The solution was extracted withchloroform (150 ml×4). The organic layer was dried with anhydrous sodiumsulfate, and the solvent was removed by distillation under reducedpressure. The residue was purified by recrystallization from ethanol.The crystals were dried under reduced pressure to obtain 24.0 mg (0.0558mmol, 8%) of the title compound as a pale yellow powder.

mp: 200-203° C.

¹H-NMR (400 MHz, 0.1N NaOD) δ ppm: 1.05 (3H, d, J=6.9 Hz), 1.21-1.33(1H, m), 1.61 (1H, dt, J=25.2, 7.6 Hz), 2.30-2.40 (1H, m), 2.51 (3H, s),3.22 (1H, d, J=10.3 Hz), 3.48 (1H, t, J=8.6 Hz), 3.54-3.61 (1H, m), 3.94(1H, dd, J=10.5, 1.5 Hz), 4.06-4.12 (1H, m), 4.51 (2H, d, J=47.4 Hz),5.01 (1H, ddd, J=64.2, 9.1, 5.1 Hz), 7.69 (1H, d, J=14.5 Hz), 8.45 (1H,d, J=3.7 Hz).

Elementary analysis for C₂₀H₂₂F₃N₃O₃.0.25EtOH.0.5H₂O:

Calculated: C, 57.27; H, 5.74; N, 9.77.

Found: C, 57.17; H, 5.74; N, 9.56.

MS (ESI) m/z: 410 (M+H)⁺.

IR (ATR) ν: 3400, 3367, 3089, 2964, 2883, 1711, 1618, 1508, 1468, 1435,1356, 1321, 1259, 1227, 1178, 1130 cm⁻¹.

Reference Example 102 Tertiary butyl5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate

To a suspension of 5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylicacid produced by the method described in Culbertson T. P., Domagala J.M., Nichols J. F., Priebe S., and Skeean R. W., J. Med. Chem., 1987, 30,1711-1715 (1165 g, 4.994 mol) in dichloromethane (10 L), O-tertiarybutyl-N,N′-diisopropylurea (3020 g, 15.00 mol) was added at roomtemperature with stirring. When increase in the inner temperature andstarting of refluxing were noted, the reaction system was cooled in anice bath, After cooling the reaction mixture to room temperature, themixture was stirred for 1 hour after removing the ice bath, and foranother 3 hours while heating to 40° C. After stirring the reactionmixture for another 1 hour with cooling in an ice bath, the insolublecontent was removed, and the filtrate was dried under reduced pressure.The residue was purified by silica gel column chromatography (silicagel: 4 kg; elusion by hexane:ethyl acetate, 3:1) to obtain 925.2 g (64%)of the title compound (a mixture of isomers at position 3) as a paleyellow syrup. Although separation of the diastereomers of position 3 ofthe pyrrolidine was easy, the diastereomers were used without separationsince the subsequent step involved epimerization. ¹H-NMR spectrum of theisomers aliquoted for evaluation purpose are shown below.

Low Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.45 (9H, s), 1.54 (3H, d, J=7.08 Hz),2.59-2.74 (2H, m), 2.95-3.03 (1H, m), 3.14 (1H, dd, J=9.77, 8.79 Hz),3.49 (1H, dd, J=9.77, 6.35 Hz), 7.26-7.36 (5H, m).

High Polarity Isomer:

¹H-NMR (400 MHz, CDCl₃) δ: 1.36 (9H, s), 1.53 (3H, d, J=7.32 Hz),2.59-2.75 (2H, m), 3.02-3.11 (1H, m), 3.16 (1H, dd, J=10.01, 5.62 Hz),3.51 (1H, dd, J=10.01, 8.54 Hz), 7.24-7.36 (5H, m).

Reference Example 103 Tertiary Butyl(3S)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylic Acid

To a solution of 5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylicacid tertiary butyl (30.05 g, 0.104 mol) in N,N′-dimethylformamide (210mL), iodomethane 26.0 mL (59.28 g, 0.418 mol), and then, sodium hydride(55% in oil, 11.35 g, 0.260 mol) was added with stirring at roomtemperature in nitrogen atmosphere. When the inner temperature increasedto about 50° C., the reaction mixture was cooled to 30° C. by using anice bath, and then, the mixture was stirred for 23 hours by replacingthe ice bath with a water bath at an exterior temperature of 17° C. Thereaction mixture was poured to cool aqueous solution of citric acid (amixture of 1 L of 10% citric acid and 500 g of ice), and the mixture wasstirred for 30 minutes, and extracted with ethyl acetate (800 mL, 500mL). The organic layers were combined, and washed with saturated aqueoussolution of sodium chloride and dried with anhydrous sodium sulfate.After filtration, the filtrate was dried under reduced pressure. Theresidue was flash purified by silica gel column chromatography (elusionby hexane ethyl acetate, 5:1→4:1) to obtain 10.63 g (33.7%) of highpolarity isomer of the title compound as a white solid. 14.91 g (47.3%)of low polarity isomer of tertiary butyl(3R)-3-methyl-5-oxo-1-[(1R)-1-phenylethyl]pyrrolidine-3-carboxylate wasalso obtained.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.34 (12H, s), 1.52 (3H, d, J=7.10 Hz),2.27 (1H, d, J=17.0 Hz), 2.93 (1H, d, J=17.0 Hz), 3.05 (1H, d, J=10.1Hz), 3.32 (1H, d, J=10.1 Hz), 5.50 (1H, q, J=7.1 Hz), 7.23-7.38 (5H, m).

Reference Example 104 Tertiary butyl (3S)-4-[2-(tertiary butyldimethylsilyl)hydroxyethyl]-3-methyl-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylate

To a solution of(3S)-3-methyl-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylic acidtertiary butyl (30.0 g, 98.9 mmol) and tertiarybutyl(2-iodoethoxy)dimethylsilane (36.8 g, 129 mmol) in anhydroustetrahydrofuran (288 mL), lithium bis(trimethylsilyl)amide (1.0Msolution in tetrahydrofuran, 129 mL, 129 mmol) was added dropwise at −4°C., and the mixture was stirred at 2° C. for 3.5 hours. Saturatedaqueous solution of ammonium chloride (300 mL) was added to the reactionmixture, and the mixture was extracted with ethyl acetate (300 mL, 200mL). The organic layer was washed with saturated aqueous solution ofsodium chloride (200 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was dried under reduced pressure to obtain 54.1g of the title compound. The product was used in the subsequent stepwith no further purification.

MS (ESI) m/z: 363 (M-Boc+H)⁺.

Reference Example 105 Tertiary butyl(3S)-4-(2-hydroxyethyl)-3-methyl-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylate

The crude product in the form of a silyl compound (54.1 g, 98.9 mmol)was dissolved in tetrahydrofuran (450 mL), and 1.0 mol/l solution oftetrabutylammonium fluoride in tetrahydrofuran (148 mL, 148 mmol) wasadded dropwise to the solution in an ice bath. The mixture was stirredat room temperature for 2 hours, and after concentrating the mixture,the concentrate was extracted with ethyl acetate (200 mL, 100 mL). Theorganic layer was washed with 10% aqueous solution of sodiumhydrogencarbonate (200 mL), aqueous solution of citric acid (300 mL),and saturated aqueous solution of sodium chloride (100 mL), and driedwith anhydrous sodium sulfate. After filtration, the filtrate was driedunder reduced pressure. The residue was purified by silica gel columnchromatography (elusion by hexane:ethyl acetate, 6:1→4:1→1:1) to obtain29.1 g (83.9 mmol, 85%) of the title compound as a colorless transparentsyrup substance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 1.28 (3H, s), 1.40 (9H, s), 1.51-1.53(1H, m), 1.53 (3H, d, J=7.1 Hz), 1.78-1.94 (2H, m), 2.90-3.08 (2H, m),3.67-3.75 (1H, m), 3.80-3.91 (1H, m), 4.85-4.89 (1H, m), 5.43-5.53 (1H,m), 7.27-7.37 (5H, m).

MS (ESI) m/z: 348 (M+H)⁺.

Reference Example 106 Tertiary butyl(3S)-4-[2-(benzenesulfonyl)oxyethyl]-3-methyl-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylate

To a solution of(3S)-4-(2-hydroxyethyl)-3-methyl-5-oxo-1-[(1R)-phenylethyl]pyrrolidine-3-carboxylicacid tertiary butyl (29.1 g, 83.9 mmol) in dichloromethane (280 mL),triethylamine (15.2 mL, 109 mmol), benzenesulfonyl chloride (11.8 mL,92.3 mmol), and 4-dimethyl aminopyridine (1.02 g, 8.39 mmol) were addedin an ice bath, and the mixture was stirred at room temperature for 19hours. Saturated aqueous solution of ammonium chloride (280 mL) wasadded to the reaction mixture, and after separating the organic layerand removing the solvent under reduced pressure, the residue wasdissolved in ethyl acetate (280 mL, 180 mL). The solution was washedagain with the saturated aqueous solution of ammonium chloride. Theorganic layer was washed with 1 mol/l hydrochloric acid aqueous solution(250 mL), saturated sodium bicarbonate water (250 mL), and saturatedaqueous solution of sodium chloride (200 mL), and dried with anhydroussodium sulfate. After the filtration, the filtrate was dried underreduced pressure to obtain the crude product in the form of abenzenesulfonyl compound (43.7 g). The product was used in thesubsequent step with no further purification.

MS (ESI) m/z: 510 (M+Na)⁺.

Reference Example 107 Tertiary butyl(7S)-7-methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane-7-carboxylate

To a solution of the crude product in the form of a benzenesulfonylcompound (43.7 g, 83.9 mmol) in anhydrous tetrahydrofuran (470 mL) wasadded 1.0 mol/l solution of sodium bis(trimethylsilyl)amide intetrahydrofuran (109 mL, 109 mmol) in an ice bath, and the mixture wasstirred at room temperature for 1 hour. To the reaction mixture wasadded saturated aqueous solution of ammonium chloride (300 mL), and themixture was extracted with ethyl acetate (300 mL, 200 mL), and theorganic layer was washed with saturated aqueous solution of sodiumchloride (200 mL). After drying the organic layer with anhydrous sodiumsulfate, the residue was filtered and the filtrate was dried underpressure. The residue was purified by silica gel column chromatography(eluted with hexane:ethyl acetate, 3:1→2:1) to obtain 24.6 g (89%, 2steps) of the title compound as a white solid.

mp: 55-57° C.

[α]_(D) ^(25.1)=122.1° (c=0.517, CHCl₃).

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.72-0.77 (1H, m), 0.85-0.90 (1H, m),1.04-1.13 (2H, m), 1.18 (3H, s), 1.32 (9H, s), 1.54 (3H, d, J=7.1 Hz),3.08 (1H, d, J=9.8 Hz), 3.53 (1H, d, J=9.8 Hz), 5.52 (1H, q, J=7.1 Hz),7.26-7.34 (5H, m).

Elementary analysis for C₂₀H₂₇NO₃:

Calculated: C, 72.92; H, 8.26; N, 4.25.

Found: C, 72.64; H, 8.27; N, 4.06.

MS (FAB) m/z: 330 (M+H)⁺.

HRMS (FAB) m/z: 330.2069 (Calcd for C₂₀H₂₈NO₃ 330. 2069).

IR (ATR) ν: 3066, 2976, 2933, 2879, 1720, 1676, 1481, 1454, 1433, 1365,1329, 1286, 1238, 1203 cm⁻¹.

X ray structural analysis was conducted to determine the configurationof position 7 of this compound. The results were as shown in FIG. 3.

After collecting the data, initial phase was resolved by direct method,and refined by full matrix least square method. In the refinement,anisotropic thermal parameters were used for the non-hydrogen atoms, andhydrogen atoms were placed in calculated positions in the coordinates.This compound has two asymmetric carbon atoms, and absoluteconfiguration of one asymmetric carbon atom was known. The absoluteconfiguration of the other asymmetric carbon atom, therefore, wasdetermined based on the absolute configuration of the known asymmetriccarbon atom. The results are shown in FIG. 1. The configuration of theposition 7 of the title compound was thus determined to be (S). Theconfiguration of a series of compounds produced by using this compoundas an intermediate was also determined.

Reference Example 108(7S)-7-Methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane-7-carboxylicAcid

To a solution of tertiary butyl(7S)-7-methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane-7-carboxylate(24.5 g, 74.4 mmol) in dichloromethane (120 mL), trifluoroacetic acid(120 mL) was added dropwise in an ice bath, and the mixture was stirredfor 2 hours. The reaction mixture was dried under reduced pressure, andafter adding toluene (20 mL) to the residue, the mixture was dried underreduced pressure. The residue was dissolved in 1 mol/l aqueous solutionof sodium hydroxide (300 mL), and the aqueous solution was washed withethyl acetate (350 mL). To the aqueous layer was added concentratedhydrochloric acid (25 mL) to pH 2 to 3 in an ice bath, and the mixturewas extracted with chloroform (300 mL×2). The organic layer was washedwith water (200 mL) and saturated aqueous solution of sodium chloride(100 mL), and dried with anhydrous sodium sulfate, and the solvent wasremoved by distillation under reduced pressure. Toluene (20 mL) wasadded to the residue, and the mixture was dried under reduced pressure.The residue was suspended in chloroform (20 mL), and hexane (200 mL) wasadded for recrystallization. The precipitated solid was washed withhexane (100 mL), and dried under reduced pressure to obtain 20.48 g(quantitative) of the title compound as a white solid. The product wasused in the subsequent step with no further purification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.78-0.83 (1H, m), 0.90-0.95 (1H, m),1.08-1.18 (2H, m), 1.24 (3H, s), 1.55 (3H, d, J=7.3 Hz), 3.11 (1H, d,J=10.0 Hz), 3.55 (1H, d, J=10.0 Hz), 5.52 (1H, q, J=7.1 Hz), 7.28-7.32(5H, m).

MS (ESI) m/z: 274 (M+H)⁺.

Reference Example 109(7S)-7-Amino-7-methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane

To a solution of(7S)-7-methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane-7-carboxylicacid (20.4 g, 74.4 mmol) and diphenylphosphoric acid azide (17.6 mL,81.8 mmol) in toluene (200 mL), triethylamine (20.7 mL, 149 mmol) wasadded, and the mixture was stirred in an oil bath at 125° C. for 1 hour.The reaction mixture was concentrated under reduced pressure to obtainthe crude product in the form of an isocyanate.

After dissolving the crude product in the form of an isocyanate in1,4-dioxane (180 mL), water (90 mL) and concentrated hydrochloric acid(90 mL) were added to the mixture. The mixture was stirred in an oilbath at 50° C. for 1 hour, and water (200 mL) was added to the reactionmixture. After washing with ethyl acetate (200 mL), 10 mol/l aqueoussolution of sodium hydroxide (170 mL) was added to the aqueous layer topH 9 to 10, and the solution was extracted with toluene (200 mL×2). Theorganic layer was washed with saturated aqueous solution of sodiumchloride (100 mL), and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure toobtain 15.8 g (64.7 mmol) of the title compound as a pale yellow oilyproduct. The product was used in the subsequent step with no furtherpurification.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.72-0.78 (2H, m), 0.99-1.10 (2H, m),1.08 (3H, s), 1.53 (3H, d, J=7.4 Hz) 2.82 (1H, d, J=9.6 Hz), 3.27 (1H,d, J=9.6 Hz), 5.56 (1H, q J=7.1 Hz), 7.14-7.37 (5H, m).

Reference Example 110 (7S)-7-(Tertiarybutoxycarbonylamino)-7-methyl-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane

The(7S)-7-amino-7-methyl-4-oxo-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane(15.8 g, 64.7 mmol) was dissolved in toluene (82 mL), and a solution of65% (by weight) solution of sodium bis(2-methoxyethoxy)aluminum hydridein toluene solution (77.6 mL, 259 mmol) in toluene (6 mL) was addeddropwise in 15 minutes in an ice bath so that the inner temperature didnot exceed 70° C., and the mixture was stirred at room temperature for10 minutes. The reaction mixture was cooled in an ice bath, and 25% (byweight) aqueous solution of sodium hydroxide (100 mL) was addeddropwise. After quenching the solution, the solution was extracted withtoluene (135 mL). The organic layer was washed with saturated aqueoussolution of sodium chloride (100 mL), and di-tert-butyl dicarbonate(15.6 g, 71.2 mmol) was added. The reaction mixture was stirred at roomtemperature for 3 hours, and the solvent was removed by distillationunder reduced pressure. The residue was purified by silica gel columnchromatography (elusion by hexane ethyl acetate, 8:1→4:1→1:1) to obtain18.0 g (73%) of the title compound as a colorless transparent syrupsubstance.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.37-0.49 (2H, m), 0.62-0.68 (1H, m),0.77-0.82 (1H, m), 1.20 (3H, s), 1.32 (3H, d, J=6.6 Hz), 1.44 (9H, s),2.46 (2H, dd, J=33.2, 9.3 Hz), 2.68 (1H, d, J=8.8 Hz), 3.27 (1H, q,J=6.6 Hz), 3.31-3.34 (1H, m), 4.71 (1H, s), 7.19-7.34 (5H, m).

MS (ESI) m/z: 331 (M+H)⁺.

Reference Example 111 (7S)-7-(Tertiarybutoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane

To a solution of (7S)-7-(tertiarybutoxycarbonylamino)-7-methyl-5-[(1R)-phenylethyl]-5-azaspiro[2.4]heptane(18.0 g, 54.5 mmol) in methanol (180 mL) was added 10% palladium-carboncatalyst (water content, 52.8%; 9.00 g), and the mixture was stirred atroom temperature for 18 hours in hydrogen gas atmosphere, and in an oilbath at 40° C. for 5.5 hours. After removing the catalyst, the solventwas dried under reduced pressure to obtain 13.4 g (quantitative) of thecrude target compound as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ ppm: 0.38-0.43 (1H, m), 0.54-0.61 (2H, m),0.74-0.80 (1H, m), 1.08 (3H, s), 1.44 (9H, s), 2.75 (1H, d, J=7.6 Hz),2.78 (1H, d, J=7.1 Hz), 3.13 (1H, d, J=11.5 Hz), 3.73-3.77 (1H, m), 4.45(1H, s).

MS (ESI) m/z: 227 (M+H)⁺.

Reference Example 1127-[(7S)-7-Amino-7-methyl-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicAcid

(7S)-7-(tertiary butoxycarbonylamino)-7-methyl-5-azaspiro[2.4]heptane(13.4 g, 54.5 mmol),6,7-difluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid—difluoroborane complex (17.9 g, 49.5 mmol), and triethylamine (8.97mL, 64.4 mmol) were dissolved in dimethyl sulfoxide (52 mL), and themixture was stirred in an oil bath at 40° C. for 17 hours. The reactionmixture was poured into cold water (1000 mL), and the precipitated solidwas collected by filtration. To this solid were added a mixed solutionof ethanol and water (ethanol:water, 5:1) (180 mL) and triethylamine (15mL), and the mixture was heated under reflux for 1.5 hours. The reactionmixture was dried under reduced pressure, and the residue was dissolvedin ethyl acetate (150 mL×2) and washed with 10% aqueous solution ofcitric acid (200 mL), water (200 mL), and saturated aqueous solution ofsodium chloride (100 mL). The organic layer was dried with anhydroussodium sulfate, and the solvent was removed by distillation underreduced pressure. The residue was dissolved in a mixed solution ofchloroform and methanol (chloroform:methanol, 9:1) (100 mL), and afteradding silica gel (10 g), the mixture was stirred for 1 hour. Afterremoving silica gel by filtration, the mixture was washed with a mixedsolution of chloroform and methanol (chloroform:methanol, 9:1) (50mL×2), and the filtrates were combined and concentrated to dryness. Theresidue was dissolved in concentrated hydrochloric acid (200 mL) in anice bath, and stirred at room temperature for 30 minutes. The reactionmixture was washed with chloroform (400 mL×5). In an ice bath, 10 mol/laqueous solution of sodium hydroxide was added to the aqueous layer toadjust the pH to 11.8, and the pH was further adjusted to 7.4 by addinghydrochloric acid. The solution was extracted by adding chloroform (1000mL×3). The organic layer was dried with anhydrous sodium sulfate, andthe solvent was removed by distillation under reduced pressure. Theresidue was purified by recrystallization from ethanol, and the crystalswere dried under reduced pressure to obtain 18.5 g (79%) of the titlecompound as a pale pink powder.

¹H-NMR and other data from instrumental analysis of this product werefully consistent with the data of the compound produced in Example 9. Inother words, of the quinolone derivatives having7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl group, the quinolonederivative described in Example 9 which is a compound with high activityhas a 5-azaspiro[2.4]heptane-5-yl group in which the stereochemicalconfiguration at position 7 is (7S).

Test Example 1

The compounds of the present invention were evaluated for theirantibacterial activity according to the standard method defined byJapanese Society of Chemotherapy, and the results are shown in MIC(μg/ml) in the table, below. In the table, MIC value is also shown formoxifloxacin (MFLX), Comparative compound 1 which is the compounddescribed in Japanese Patent Application Laid-Open No. 2-231475 (PatentDocument 2), levofloxacin (LVFX), gatifloxacin (GTFX), and ciprofloxacin(CPFX), in addition to the compound of the present invention. (Thestructure below shows only the core structure). S. aureus, 87037 isLVFX-resistant MRSA and S. pneumoniae, J24 is penicillin-intermediateresistant bacteria.

TABLE 1 Example 1 Example 2 Example 3 Example 8 Example 9 Example 10Example 15 Example 16 E. coli NIHJ 0.025 0.012 0.012 0.012 0.025 0.0250.012 0.012 P. vulgaris, 08601 0.05 0.025 0.05 0.025 0.025 0.05 0.0250.006 S. marscecens, 10100 0.2 0.1 0.2 0.2 0.1 0.2 0.2 0.1 P.aeruginosa, 32104 0.39 0.2 0.39 0.78 0.39 0.39 0.39 0.1 P. aeruginosa,32121 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.05 S. aureus, 209P 0.025 0.025 0.0250.025 0.025 0.05 0.05 0.025 S. epidermidis, 56500 0.1 0.05 0.1 0.1 0.10.2 0.1 0.1 E. faecalis, ATCC 19433 0.2 0.2 0.2 0.2 0.2 0.78 0.2 0.39 S.aureus, 87037 0.78 0.78 0.78 0.78 0.78 1.56 0.78 3.13 S. pneumoniae, J240.1 0.1 0.1 0.1 0.1 0.39 0.1 0.2 Example 17 Example 21 Example 22Example 23 Example 27 Example 28 Example 29 Example 30 E. coli NIHJ0.006 0.012 0.012 ≦0.003 0.006 0.025 0.05 0.006 P. vulgaris, 08601 0.0120.05 0.012 0.006 0.006 0.05 0.025 0.025 S. marscecens, 10100 0.1 0.2 0.10.05 0.1 0.39 0.2 0.1 P. aeruginosa, 32104 0.39 0.78 0.39 — 0.39 — 0.20.2 P. aeruginosa, 32121 0.1 0.2 0.1 0.05 0.1 0.39 0.1 0.05 S. aureus,209P 0.025 0.025 0.025 0.025 0.012 0.025 0.05 0.05 S. epidermidis, 565000.1 0.1 0.05 0.05 0.05 0.05 0.2 0.2 E. faecalis, ATCC 19433 0.2 0.2 0.20.1 0.2 0.2 0.78 0.39 S. aureus, 87037 0.39 0.78 0.78 0.39 0.39 0.2 3.131.56 S. pneumoniae, J24 0.1 0.1 0.1 0.05 0.1 0.1 0.39 0.2 Example 31MFLX Cop. Ex. 1 LVFX GTFX CPFX E. coli NIHJ 0.006 0.012 ≦0.003 0.0120.006 ≦0.003 P. vulgaris, 08601 0.012 0.025 0.012 0.012 0.006 ≦0.003 S.marscecens, 10100 0.1 0.1 0.05 0.1 0.1 0.025 P. aeruginosa, 32104 — 0.390.1 0.2 0.2 0.05 P. aeruginosa, 32121 0.05 0.2 0.05 0.1 0.1 0.025 S.aureus, 209P 0.012 0.05 0.006 0.2 0.05 0.1 S. epidermidis, 56500 0.0250.2 0.05 0.39 0.2 0.2 E. faecalis, ATCC 19433 0.1 0.2 0.1 0.78 0.39 0.78S. aureus, 87037 0.1 1.56 0.39 >6.25 1.56 >6.25 S. pneumoniae, J24 0.050.1 0.025 0.78 0.2 0.39

Test Example 2

The compounds produced in Examples 2, 3, and 9 of the present inventionwere evaluated by mouse bone marrow micronucleus test by using 5 animalsper group of 6-week-old Slc:ddY male mice, and the compounds dilutedwith 0.1 mol/l NaOH/physiological saline. The control was 0.1 mol/lNaOH/physiological saline solvent, and the positive control wascyclophosphamide (cyclophoshamide, CP) dissolved and diluted inphysiological saline. All samples were sterilized by filtering throughMilex GS filter (0.22 μm). Each solution was intravenously administeredat a dose of 10 mL/kg in single dose at an administration rate of 0.2mL/min.

Bone marrow cells were collected from thigh bone at 24 hours after theadministration, and smear preparation was prepared. After staining withacridine orange, 1000 polychromatic erythrocytes per animal wereobserved under fluorescence microscope to count frequency ofmicronucleated polychromatic erythrocyte and ratio of the orthochromaticerythrocytes to the polychromatic erythrocyte in 1000 erythrocytes.

No significant difference in the micronucleus induction rate with thecontrol was found in all of the administration groups of the compound ofExample 2 at a dose of 50 and 100 mg/kg, the administration groups ofthe compound of Example 3: at a dose of 100 and 150 mg/kg, and theadministration group of the compound of Example 9 at a dose of 50, 100,and 150 mg/kg, and the evaluation result was negative. In other words,the compounds of the present invention has very weak micronucleusinduction in the in vivo mouse bone marrow micronucleus test used inevaluating genotoxicity, and therefore, highly safe.

Test Example 3

The compounds described in Examples 2, 3, and 9 of the present inventionwere evaluated for concentration in blood and other organs after theadministration by the procedure as described below. The Comparativecompound was also evaluated by the same procedure.

The test substance was orally administered to fasted rats (7 week oldmale Crj:CD IGS rats purchased from Charles River Laboratories Japan,Inc.) at a dose of 5 mg/kg.

Animals of absorption test group (3 animals per group) were sacrificedby bleeding under etherization at 0.25, 0.5, 1, 2, 4, and 8 hours afterthe drug administration, and blood, lever, kidney, and lung werecollected. The blood was centrifuged (3000 rpm×15 minutes, 4° C.) aftercoagulation to collect serum. Tissue was homogenized after adding 3 to 5mL of 0.1 mol/l phosphate buffer (pH 7.0), and the supernatant wascollected from the homogenate (3000 rpm×15 minutes, 4° C.).

Animals of excretion test group (3 animals per group) were placed in ametabolic cage, and the urine of 0 to 4 hours and 4 to 24 hours afterthe administration was collected in a water-cooled container.Simultaneously with the collection of the urine, the cage was alsowashed with about 15 mL of 0.1 mol/l phosphate buffer (pH 7.0) tocollect the urine attached to the cage. For evaluation of conjugatessuch as glucuronide, the collected sample was also aliquoted andhydrolyzed with an equal amount of 1 mol/l aqueous solution of sodiumhydroxide, and the hydrolysate was neutralized with 0.5 mol/lhydrochloric acid and measured for its concentration. The concentrationwas measured by LC-MS/MS method.

Pharmacokinetic parameters of the each drug in rat were calculated byfrom the time course of the average concentration by usingpharmacokinetic analysis software PSAG-CP (AS Medica Inc.) in a mannernot dependent on the model animal.

TABLE 2 Pharmacokinetics in rat Compound Comparative Example 2 Example 3Example 9 compound 1 MFLX Hydrating molecule 0.75H₂O 2H₂O EtOH•0.5H₂OHCl•H₂O HCl•0.5H₂O Serum Cmax (μg/mL) (0.25 h) 0.67 1.03 1.22 0.82 1.49AUC0-8 h (μg · h/mL) 1.31 1.85 3.08 1.59 4.46 Tissue Cmax (μg/g) Liver10.2 9.02 13.7 9.3 9.49 Kidney 7.78 8.83 9.88 8.63 10.1 Lung 2.46 3.473.86 2.59 4.69 AUC0-8 h (μg · h/mL) Liver 17.4 15.4 32.1 15.0 — Kidney14.7 17.1 27.8 19.3 — Lung 8.07 7.38 13.7 6.77 — Recovery (%) in urinein relation 0-24 h 25.4 24.5 27.7 21.8 26.6 to the amount administeredRecovery (%) including 26.6 24.7 32.0 23.1 30.8 conjugate

By repeating the procedure used for the rats, the compound of Example 9,comparative compound 1, and MFLX were evaluated for cynomolgus monkey byusing fasted female cynomolgus monkey (3 animals per group) which hadbeen administered with a dose of 5 mg/kg in single dose, and measuringintact compounds in serum and excreted urine. The measurement wasconducted by LC-MS/MS method.

TABLE 3 Pharmacokinetics in cynomolgus monkey Compound ComparativeExample 9 compound 1 MFLX Hydrating molecule EtOH•0.5H₂O HCI•H₂OHCI•0.5H₂O Serum Cmax (μg/mL) 2.18 0.84 1.03 t½ (h) 4.8 5.0 5.3 AUC0-24h 16.9 5.11 6.64 (μg · h/mL) Recovery (%) in urine 0-24 h 61.3 25.8 8.1in relation to the amount Recovery (%) 59.5 32.9 12.2 administeredincluding conjugate

As apparent from the data of the serum concentration, the tissueconcentration, and AUC, the compound of the present invention, and inparticular, the compound of Example 9 exhibited a serum concentrationand a tissue concentration by oral administration which is about 2 timeshigher, and an AUC value which is 1.5 to 2 times higher than those ofthe Comparative compound 1 indicating the excellent oral absorptivityand tissue penetration of the present compound. Excretion rate in urinewas about 1.5 times higher than that of the Comparative compound 1indicating the excellent urine excretion. The data was even superior incynomolgus monkey, and the blood penetration was about 2.5 times higher,and the urine excretion rate was more than 2 times higher than those ofthe Comparative compound 1.

The compound of Example 9 and MFLX exhibited similar pharmacokinetics inrat and cynomolgus monkey. However, the compound of Example 9 exhibitedsignificantly superior blood penetration and urine excretion incynomolgus monkey, clearly indicating that the compound of Example 9exhibits excellent pharmacokinetic properties not only in single speciesbut in more than one animal species.

Test Example 4

The potential convulsant activity upon intracisternal administration tomouse was evaluated according to the method of Ueda et al. (Eur. J.Pharmacol., 1979, 56, 265-268). The test substance was intracisternallyadministered to male Slc:ddy mice animals per group), and the convulsionand death were monitored for each cage until 30 minutes after theadministration. The test substance was dissolved in 5 μl of 1% aqueoussolution of lactic acid, and the dose was 5, 15, or 50 μg per animal.

TABLE 4 Number of mice which showed convulsion Example 2 Example 3Example 9 Comparative compound 1 MFLX CPFX  5 μg/mouse 1/6 0/6 0/6 0/60/6 1/6 15 μg/mouse 4/6 2/6 2/6 0/6 0/6 3/6 50 μg/mouse 6/6 6/6 6/6 0/60/6 6/6

The potential convulsant activity upon intracisternal administration tomouse was also evaluated for the concomitant and administration with4-biphenyl acetic acid (BPAA: an active metabolite of fenbufen) and inthe absence of such concomitant administration. The evaluation wasconducted by using 4 week old male Slc:ddY mice (6 animals per group)and intracisternally administering the test substance at a dose of 5μg/5 μl/mouse (solvent, 0.5% lactic acid). Monitoring of the animals forthe convulsion and death was started immediately after theadministration and continued to 30 minutes after the administration.When used concomitantly with the BPAA, BPAA was suspended in 5% CMC, and400 mg/kg was orally administered at a solution amount of 10 mL/kg, andthe test substance was intracisternally administered 30 minutes afterthe BPAA.

TABLE 5 Effect of biphenyl acetate Dose Number of animals which showedconvulsion μg/5 μL/mouse, (number of dead animals) i. cist BPAA

BPAA

0.5% lactate 0 0/6 (0/6) 0/6 (0/6) Compound of Example 9 5 0/6 (0/6) 0/6(0/6) CPFX 5 0/6 (0/6) 6/6**## (6/6**##) *p < 0.05, **p < 0.01:Sinificantly different from 0.5% Lactate group (Fisher's probabilitytest) ^(#)p < 0.05, ^(##)p < 0.01: Sinificantly different from thewithout BPAA group (Fisher's probability test)

The compound of the present invention exhibited a convulsion inducingfrequency at a high dose which is higher that of the comparativecompound 1 but lower than ciprofloxacin (CPFX) which are widely used inclinical practice indicating weaker convulsion inducing activity, hence,higher safety compared to CPFX. The test of concomitant administrationwith the biphenyl acetic acid which is the model of the concomitantadministration with the fenbufen also indicated the excellent safety ofthe present compound since no case of convulsion or death was found forthe compound of Example 9 whereas convulsion and death were noted in theadministration of ciprofloxacin.

Test Example 5

Guinea pig maximization test (GPMT) which is a widely accepted model fordelayed antigenicity was conducted according to the method of Mugnussonet al. (J. invest. Dermatol., 52, 1969) by using a cutaneoussensitization concentration of 1% and a patch sensitization andinduction concentration of 10%. At day 1, the animal was sensitized bycutaneously administering the test substance (the quinolone compoundsand the control: vehicle, vaseline) (1% solution in physiologicalsaline + FCA emulsion) at the back of the head of the shaved guinea pigs(7 week old male Slc:Hertley). At day 7, lauryl sodium sulfate (SLC) wasapplied for stimulus (adjuvant treatment), and on the next day, the testsubstance coated on a wax paper was patched onto the shaved skin to forsealed sensitization, and after 48 hours (at day 10), the waxed paperwas removed. The skin reaction was observed upon the removal. On day 22,the test substance (10%) was patched onto the anterior side of the bodyfor induction, and the induction patching was removed after 24 hours. Onthe next day (at day 24) and the next day (at day 25), the skin reactionwas evaluated according to the description of the document as mentionedabove. Erythema and edema were scored, and the case with the total scoreof 2 or higher was evaluated to be positive with the maximum of thescore being 7.

TABLE 6 Average score and the results of evaluation in GPMT Example 9Comparative compound 1 MFLX Gemifloxacin Averate score 0 6.8 0 6.8Evaluation Positive Positive Negative Positive

The compound of Example 9 was confirmed to be GPMT negative (score, 0).On the other hand, Comparative compound 1 exhibited a score of 6.8,which is almost the highest score. In the meanwhile, gemifloxacin(gemifloxacin mesylate; product name, FACTIVE™) which recently becamecommercially available in the U.S. has been reported in the phase 3clinical trial which was conducted for community-acquired pneumonia andacute exacerbation of chromic bronchitis that it frequently develops theside effect of rash and the rash development is frequently found afterday 7 of the repeated administration. This gemifloxacin was alsoconfirmed to be GPMT positive with the score of 6.8 which is almost thehighest score as was the case Comparative compound 1. Since thegemifloxacin that had been reported to induce rash development was GPMTpositive, the GPMT negative compound of the present invention wasindicated to have a reduced risk of rash development.

Test Example 6

Measurement of hERG-K⁺ channel blocking effect which is an in vitrostandard evaluation system for cardiotoxicity (an abnormality inducinglethal arrhythmia which is found by an electrocardiogram and which isobserved as prolonged QT or QTc interval) which has recently beenreported as a side effect of quinolone antibacterial agent was conductedby the method described in Biophysical Journal, vol. 74, page 230, 1998.

TABLE 7 hERG-K⁺ channel blocking effect (1) Inhibition Comparative (%)Example 2 Example 3 Example 9 compound 1 MFLX GTFX  30 μM 3.8 ± 2.1  5.3± 4.1  7.4 ± 4.5 6.7 ± 8.3 33.9 ± 8.3 8.1 ± 6.0 100 μM 8.5 ± 1.2 12.0 ±4.0 10.2 ± 5.4 13.5 ± 3.1   44.6 ± 10.2 17.6 ± 11.5 300 μM 18.2 ± 4.2 22.3 ± 0.7 20.1 ± 7.4 24.8 ± 16.5 69.9 ± 8.3 39.0 ± 10.8 n = 3

TABLE 8 hERG-K⁺ channel blocking effect (2) Inhibition (%) Example 8Example 22 Example 20 Example 25 Example 23 Example 27  30 μM 1.5 ± 1.8 −1.3 ± 10.1 1.4 ± 4.5 2.0 ± 7.2 3.0 ± 4.1 −1.8 ± 6.8  100 μM 2.1 ± 2.3−0.6 ± 7.9 8.5 ± 6.1 4.2 ± 7.9 4.6 ± 4.7 0.6 ± 8.0 300 μM 2.3 ± 7.3 21.5± 6.9 14.5 ± 6.0  17.1 ± 14.4 16.7 ± 8.4   8.9 ± 11.0 n = 3

The hERG-K⁺ channel blocking effect was confirmed to be markedly weak inthe compound of the present invention compared to MFLX and GTFX withclinical reports for the action of elongating the QT or QTc interval,and Comparative compound 1.

Test Example 7

Mechanism-based inhibition (MBI) of CYP3A4 was evaluated by usinginhibition of hydroxylation at position 1 of midazolam. WhileComparative compound 1 exhibited significant inhibition in a mannerdependent on the preincubation time and the drug concentration, thecompound of Example 9 exhibited weak inhibition even when used at a highconcentration.

Several mechanisms are involved in the drug interaction by the CYPinhibition, and among such inhibition, the inhibition by generation of astable complex of the metabolite of the concomitant drug with the CYP,and the inhibition by the inactivation of the CYP by the binding of themetabolite of the concomitant drug with the hem- or apo-protein moietyare irreversible, and such irreversible inhibition may last for asubstantial period after stopping the administration of the concomitantdrug and may induce a serious side effect. Such irreversible inhibitionis called a “metabolism-based inhibition”. Of the CYP molecular speciesinvolved in the drug metabolism in human, CYP3A4 is involved in themetabolism of 50% or higher of the drug in clinical use. (Non-patentdocument: Drug Metabolism, 2nd ed., Tokyo Kagaku Dojin, 2000).Accordingly, a reagent which exhibits MBI action for CYP3A4 can beregarded as a substance having a high risk of being involved in a druginteraction.

For example, clarithromycin which is frequently used as a therapeuticdrug for bacterial respiratory infection is known to exhibit MBI actionfor CYP3A4 (see the document as mentioned above), and use ofclarithromycin concomitantly with terfenadine (an antihistamine) iscontraindicated since such concomitant administration results in theincreased blood concentration of the terfenadine due to the inhibitionof the terfenadine metabolism by CYP3A4 caused by the clarithromycin,and prolonged QT interval in the electrocardiogram, ventriculararrhythmia, and occasionally, cardiac arrest are found. However, thecompound of Example 9 was revealed to have a clearly weaker MBI evenwhen tested at a high concentration (with a significant safe margincompared to the postulated concentration in clinical use). Therefore,the compound of the present invention is estimated to be associated witha greatly reduced risk of developing clinical side effects by the druginteraction based on the MBI action for CYP3A4.

Test Example 8

Mouse local lung infection model by penicillin resistant Streptococcuspneumoniae (PRSP) was used to compare the therapeutic effect of thecompound of Example 9 and Comparative compound 1.

PRSP strain 033806 that had been anaerobically cultivated in Todd HewittBroth was nasally administered to Male CBA/JNCrlj mice (3 to 4 week old;Charles River Laboratories Japan, Inc.; 4 animals per group) underanesthesia with ketamine-xylazine mixture. Compound of Example 9 andcomparative compound 1 were orally administered to this injection model,respectively, at a dose shown in FIG. 2 (25, 50, and 100 mg/kg/day) at 2and 8 hours after the infection (treated for only 1 day at a daily doseof 50, 100, or 200 mg/kg/day). Untreated control group was administeredwith distilled water for injection.

Number of the bacteria in the lung was measured immediately before thedrug administration for the untreated group (2 hours after theinfection, indicated in the drawing as “Pre-control”), and on the nextday of the drug administration for the untreated group (the next day ofthe infection, indicated as “Post-control”) and treated groups for useas an index of therapeutic effect.

As evident from FIG. 2, while the in vivo antibacterial activity of thecompound of Example 9 for the test bacteria was about ¼ of thecomparative compound 1, no significant difference was found between thetherapeutic effect of the compound of Example 9 and that of thecomparative: compound 1 in the oral administration to the mouse locallung infection model by PRSP for all groups administered with the samedose.

Test Example 9 Therapeutic Effect in Rat Simple Cystitis Model (E. Coli)

-   Infection model: Rats (7 week old male Crl:CD(SD) (IGS) rats,    Charles River Laboratories Japan, Inc., 4 animals per group) that    had been deprived of water from the previous day were anesthetized    with ketamine-xylazine mixture, and E. coli strain E77156 was    transurethrally inoculated (1.2×10⁷ CFU/rat) in the bladder. After    the administration, urethral orifice was closed for 2 hours to    thereby prevent discharge of the bacterial solution, and feeding of    the water was started simultaneously with the termination of    closure.-   Drug administration: Compound of Example 9 and Comparative Compound    1 were orally administered respectively at a dose of 5, 20, or 80    mg/kg on the next day of the infection in a single dose.-   Evaluation of the effectiveness: Number of the bacteria in the    bladder was measured immediately before the drug administration and    on the next day of the drug administration (2 days after the    infection) for the untreated group, and on the next day of the drug    administration for the treated groups for use as an index of    therapeutic effect.-   Results: A significant decrease in the number of bacteria was found    only for the compound of Example 9 when the dose was 20 or 80    mg/kg/day. The therapeutic effect of this compound for the group    with the dose of 5 mg/kg/day was significantly stronger than the    Comparative Compound 1. Accordingly, the compound of Example 9 was    demonstrated to be a compound which is capable of realizing    therapeutic effects superior to those of the Comparative Compound 1    (FIG. 4).

Test Example 10

The compounds of the present invention were evaluated for theiranti-Mycobacterium tuberculosis activity according to the standardmethod defined by Japanese Society of Chemotherapy (Journal of JapaneseSociety of Chemotherapy, vol. 29, pages 76 to 79, 1981), and the resultsare shown in MIC (μg/ml) in the Tables 9 and 10, below. The compounds ofthe present invention exhibited superior antibacterial activity forMycobacterium tuberculosis.

TABLE 9 Anti-Mycobacterium tuberculosis (sensitive bacteria) activity(MIC: μg/ml) Comparative Strain/compound Example 9 RFP compound 1 TB-s 20.06 0.03 0.125 TB-s 3 0.06 0.125 0.125 TB-s 4 0.06 0.06 0.125 TB-s 50.06 0.06 0.06 TB-s 6 0.06 0.125 0.125 TB-s 7 0.06 0.06 0.25 TB-s 8 0.030.015 0.06 TB-s 9 0.06 0.06 0.125 TB-s 10 0.03 0.06 0.06 TB-s 11 0.060.06 0.125 TB-s 12  0.125 0.125 0.25 TB-s 13 0.06 0.06 0.125 TB-s 140.06 0.03 0.125 TB-s 15 0.06 0.06 0.125 TB-s 16 0.06 0.06 0.125 TB-s 170.06 0.06 0.125 TB-s 18 0.06 0.03 0.125 TB-s 19 0.06 0.125 0.125 TB-s 200.06 0.25 0.25 TB-s 21 0.06 0.03 0.125 TB-s 22 0.03 0.015 0.06 Kurono0.03 0.06 0.06 H37Rv 0.03 0.125 0.06 Ravenel 0.03 0.125 0.06 RFP:Rifampicin

TABLE 10 Anti-Mycobacterium tuberculosis (quinolone sensitive) activity(MIC: μg/ml) Strain/compound Example 9 RFP Comparative compound 1 S 1 116 2 MDR 1 0.125 16 0.5 MDR 3 0.125 128 0.5 MDR 4 0.25 64 0.5 MDR 5 0.532 1 MDR 7 0.125 >128 0.5 MDR 9 0.125 128 0.5 MDR 12 0.125 128 0.5 1(QR-3) 0.125 >128 1 2 (QR-6) 0.5 >128 128 3 (QR-1) 0.25 128 2 4 (QR-9)0.25 >128 32

1. A compound of formula (I):

or a salt thereof, wherein R¹ and R² each represents hydrogen atom; R³represents an alkyl group containing 1 to 6 carbon atoms, which beingoptionally substituted with halogen atom; R⁴ and R⁵ together represent a3-membered cyclic structure including the carbon atom shared by R⁴ andR⁵ to form a spirocyclic structure with the pyrrolidine ring; R⁶ and R⁷,each represents hydrogen atom; R⁸ represents a halogen-substitutedcycloalkyl group containing 3 to 6 carbon atoms; R⁹ represents hydrogenatom or an alkyl group containing 1 to 6 carbon atoms; X¹ representshydrogen atom or a halogen atom; and A represents a moiety representedby formula (II):

wherein X² represents an alkyl group containing 1 to 6 carbon atoms oran alkoxy group containing 1 to 6 carbon atoms.
 2. The compound, or asalt thereof, according to claim 1, wherein the compound represented bythe formula (I) is a compound represented by the following formula:

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, X¹, and A are as definedabove.
 3. The compound, or a salt thereof, according to claim 1, whereinR³ in formula (I) is methyl group.
 4. The compound, or a salt thereof,according to claim 1, wherein X¹ in formula (I) is fluorine atom.
 5. Thecompound, or a salt thereof, according to claim 1, wherein X² in formula(II) is methyl group or methoxy group.
 6. The compound, or a saltthereof, according to claim 1, wherein R⁸ in formula (I) is a1,2-cis-2-halogenocyclopropyl group.
 7. The compound, or a salt thereof,according to claim 1, wherein R⁸ in formula (I) is a stereochemicallypure 1,2-cis-2-halogenocyclopropyl group.
 8. The compound, or a saltthereof, according to claim 7, wherein the 1,2-cis-2-halogenocyclopropylgroup which is R⁸ in formula (I) is (1R,2S)-2-halogenocyclopropyl group.9. The compound, or a salt thereof, according to claim 8, wherein the(1R,2S)-2-halogenocyclopropyl group which is R⁸ in formula (I) is(1R,2S)-2-fluorocyclopropyl group.
 10. The compound, or a salt thereof,according to claim 1, wherein R⁹ in formula (I) is hydrogen atom. 11.The compound, or a salt thereof, according to claim 1, wherein thecompound of formula (I) is a stereochemically pure compound.
 12. Apharmaceutical composition comprising the compound according to claim 1,or a salt thereof, and a pharmaceutically acceptable carrier.
 13. Amethod for treating a bacterial or microbial infection, comprisingadministering an effective amount of the compound according to claim 1,or a salt thereof, to a subject in need thereof, wherein said bacterialor microbial infection is caused by a microorganism selected from thegroup consisting of Staphylococcus, Streptococcus pyogenes, hemolyticstreptococcus, enterococcus, pneumococcus, Peptostreptococcus, Neisseriagonorrhoeae, Escherichia coli, Citrobacter, Shigella, Klebsiellapneumoniae, Enterobacter, Serratia, Proteus, Pseudomonas aeruginosa,Haemophilus influenzae, Acinetobacter, Campylobacter, Chlamydiatrachomatis, Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium africans, Mycobacterium kansasii, Mycobacterium marianum,Mycobacterium scrofulaceum, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium xenopi, Mycobacterium fortuitum, andMycobacterium chelonae.
 14. A compound selected from the groupconsisting of:7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof; and7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylic acid, or a saltthereof.
 15. A pharmaceutical composition comprising the compoundaccording to claim 14, or a salt thereof, and a pharmaceuticallyacceptable carrier.
 16. A method for treating a bacterial or microbialinfection, comprising administering an effective amount of the compoundaccording to claim 14, or a salt thereof, to a subject in need thereof,wherein said bacterial or microbial infection is caused by amicroorganism selected from the group consisting of Staphylococcus,Streptococcus pyogenes, hemolytic streptococcus, enterococcus,pneumococcus, Peptostreptococcus, Neisseria gonorrhoeae, Escherichiacoli, Citrobacter, Shigella, Klebsiella pneumoniae, Enterobacter,Serratia, Proteus, Pseudomonas aeruginosa, Haemophilus influenzae,Acinetobacter, Campylobacter, Chlamydia trachomatis, Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africans, Mycobacteriumkansasii, Mycobacterium marianum, Mycobacterium scrofulaceum,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium xenopi,Mycobacterium fortuitum, and Mycobacterium chelonae. 17.7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof.
 18. A pharmaceutical composition comprising7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof, according to claim 17, and a pharmaceuticallyacceptable carrier.
 19. A method for treating a bacterial or microbialinfection, comprising administering an effective amount of7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methyl-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof, according to claim 17, to a subject in needthereof, wherein said bacterial or microbial infection is caused by amicroorganism selected from the group consisting of Staphylococcus,Streptococcus pyogenes, hemolytic streptococcus, enterococcus,pneumococcus, Peptostreptococcus, Neisseria gonorrhoeae, Escherichiacoli, Citrobacter, Shigella, Klebsiella pneumoniae, Enterobacter,Serratia, Proteus, Pseudomonas aeruginosa, Haemophilus influenzae,Acinetobacter, Campylobacter, Chlamydia trachomatis, Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africans, Mycobacteriumkansasii, Mycobacterium marianum, Mycobacterium scrofulaceum,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium xenopi,Mycobacterium fortuitum, and Mycobacterium chelonae. 20.7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof.
 21. A pharmaceutical composition comprising7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof, according to claim 20, and a pharmaceuticallyacceptable carrier.
 22. A method for treating a bacterial or microbialinfection, comprising administering an effective amount of7-[(7S)-7-amino-7-methyl-5-azaspiro[2.4]heptane-5-yl]-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-1,4-dihydro-4-oxoquinoline-3-carboxylicacid, or a salt thereof, according to claim 20, to a subject in needthereof, wherein said bacterial or microbial infection is caused by amicroorganism selected from the group consisting of Staphylococcus,Streptococcus pyogenes, hemolytic streptococcus, enterococcus,pneumococcus, Peptostreptococcus, Neisseria gonorrhoeae, Escherichiacoli, Citrobacter, Shigella, Klebsiella pneumoniae, Enterobacter,Serratia, Proteus, Pseudomonas aeruginosa, Haemophilus influenzae,Acinetobacter, Campylobacter, Chlamydia trachomatis, Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africans, Mycobacteriumkansasii, Mycobacterium marianum, Mycobacterium scrofulaceum,Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium xenopi,Mycobacterium fortuitum, and Mycobacterium chelonae.